Fungal strains

ABSTRACT

The present invention provides improved fungal strains. In some embodiments, the improved fungal strains find use in hydrolyzing cellulosic material to glucose.

The present application claims priority to U.S. patent application Ser.No. 14/717,447, filed May 20, 2015, which claims priority to U.S. patentapplication Ser. No. 13/286,860, filed Nov. 1, 2011, now U.S. Pat. No.9,068,235, which claims priority to U.S. Prov. Patent Appln. Ser. Nos.61/409,186, 61/409,217, 61/409,472, and 61/409,480, all of which werefiled on Nov. 2, 2010, and U.S. Prov. Patent Appln. Ser. No. 61/497,661,filed on Jun. 16, 2011, all of which are hereby incorporated byreference herein, in their entireties and for all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file CX3-082US1_ST25.TXT, created onDec. 7, 2011, 47,145 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention provides improved fungal strains. In someembodiments, the improved fungal strains find use in hydrolyzingcellulosic material to glucose.

BACKGROUND OF THE INVENTION

Cellulose is a polymer of the simple sugar glucose linked by beta-1,4glycosidic bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land-filling the materials, andlower overall greenhouse gas production. Wood, agricultural residues,herbaceous crops, and municipal solid wastes have been considered asfeedstocks for ethanol production. These materials primarily consist ofcellulose, hemicellulose, and lignin. Once the cellulose is converted toglucose, the glucose is easily fermented by yeast into ethanol.

SUMMARY OF THE INVENTION

The present invention provides improved fungal strains. In someembodiments, the improved fungal strains find use in hydrolyzingcellulosic material to glucose.

The present invention provides a fungal cell that has been geneticallymodified to reduce the amount of endogenous cellobiose dehydrogenaseactivity that is secreted by the cell, wherein the fungal cell is fromthe family Chaetomiaceae, wherein said cell comprises a deletion in thecellobiose dehydrogenase 1 (cdh1) gene. In some embodiments, the fungalcell is a species of Myceliophthora. In some further embodiments, thefungal cell is Myceliophthora thermophila. In some embodiments, thefungal cell has been genetically modified to disrupt the secretionsignal peptide of the cellobiose dehydrogenase. In some additionalembodiments, the fungal cell has been genetically modified to reduce theamount of the endogenous cellobiose dehydrogenase expressed by the cell.In some further embodiments, the fungal cell has been geneticallymodified to disrupt a translation initiation sequence in the transcriptencoding the endogenous cellobiose dehydrogenase. In some stilladditional embodiments, the fungal cell has been genetically modified tointroduce a frameshift mutation in the transcript encoding theendogenous cellobiose dehydrogenase. In some further embodiments, thefungal cell has been genetically modified to reduce the transcriptionlevel of a gene encoding the endogenous cellobiose dehydrogenase. Insome additional embodiments, the fungal cell has been geneticallymodified to disrupt the promoter of a gene encoding the endogenouscellobiose dehydrogenase. In some embodiments, the fungal cell has beengenetically modified to at least partially delete a gene encoding theendogenous cellobiose dehydrogenase. In some further embodiments, thefungal cell has been genetically modified to reduce the catalyticefficiency of the endogenous cellobiose dehydrogenase. In someadditional embodiments, one or more residues in an active site of thecellobiose dehydrogenase in the fungal cell have been geneticallymodified. In some still further embodiments, one or more residues in aheme binding domain of the cellobiose dehydrogenase in the fungal cellhave been genetically modified.

In some embodiments, the present invention provides fungal cellscomprising cellobiose dehydrogenase. In some embodiments, the cellobiosedehydrogenase comprises an amino acid sequence that is at least about85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98%,or about 99% identical to SEQ ID NO:2. In some additional embodiments,the fungal cell has been modified such that the cell secretes a reducedamount of endogenous cellobiose dehydrogenase 1 (cdhl), as compared to afungal cell prior to or without such modification.

The present invention also provides an enzyme mixture comprising two ormore cellulose hydrolyzing enzymes, wherein at least one of the two ormore cellulose hydrolyzing enzymes is expressed by the fungal cell. Insome embodiments, the enzyme mixture is a cell-free mixture. In someadditional embodiments, pretreated lignocellulose comprises at least onesubstrate of the enzyme mixture. In some further embodiments, thepretreated lignocellulose comprises lignocellulose treated by at leastone treatment method selected from acid pretreatment, ammoniapretreatment, steam explosion, and/or organic solvent extraction.

The present invention also provides methods for generating glucosecomprising contacting at least one cellulose substrate with an enzymemixture comprising two or more cellulose hydrolyzing enzymes, wherein atleast one of the two or more cellulose hydrolyzing enzymes is expressedby a fungal cell provided herein. The present invention also providesmethods for generating glucose, comprising contacting at least onecellulose substrate with at least one enzyme mixture provided herein. Insome further embodiments, the enzyme mixture is a cell-free mixture. Insome additional embodiments, the cellulose substrate is pretreatedlignocellulose. In still some further embodiments, the pretreatedlignocellulose comprises lignocellulose treated by at least onetreatment method selected from acid pretreatment, ammonia pretreatment,steam explosion, and/or organic solvent extraction In some additionalembodiments, the methods of the present invention further comprisefermenting the glucose to an end product. In some further embodiments,the end product is a fuel alcohol or a precursor industrial chemical. Insome additional embodiments, the fuel alcohol is ethanol or butanol. Instill some additional embodiments, the methods, enzyme mixtures, and/orfungal cells of the present invention provide at least one cellulosedegrading enzyme that is homologous or heterologous to the fungal cell.

The present invention also provides fermentation media comprising atleast one fungal cell and/or at least one enzyme mixture as providedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the nucleotide and amino acid sequences of M.thermophila CDH1 (SEQ ID NOS:1 and 2, respectively).

FIG. 2 provides a graph showing the relative saccharification efficiencyof CF-200 and CF-400, as measured in glucose produced from 100 g/kgglucan (pre-treated corn stover). Reactions were run at 24.6% solids,with 128 mM NaOAc, at pH 5, 55° C., 3% enzyme, in 110 μL volumes.Glucose was measured using the GOPOD assay. Error bars represent ±1 SD,n=4.

DESCRIPTION OF THE INVENTION

The present invention provides improved fungal strains. In someembodiments, the improved fungal strains find use in hydrolyzingcellulosic material to glucose. As indicated herein, the presentinvention provides improved fungal strains for the conversion ofcellulose to glucose. In particular, the improved fungal strainsprovided herein are genetically modified to reduce the amount ofendogenous cellobiose dehydrogenase activity secreted by the cells.Prior to the present invention, it was generally believed thatcellobiose dehydrogenase enhances the rate of cellulose hydrolysis byreducing the concentration of cellobiose, which is a potent inhibitor ofsome cellulase components (See e.g., Mansfield et al., Appl. Environ.Microbiol., 63:3804-3809 [1997]; and Igarishi et al., Eur. J. Biochem.,253:101-106 [1998]). Furthermore, cellobiose dehydrogenase has beenreported as playing a critical role in contributing to synergisticenhancement during cellulose degradation by preventing hydrolysisproduct inhibition (See e.g., Hai et al., J. Appl. Glycosci., 49:9-17[2002]). It was also generally believed that cellobiose dehydrogenasewas useful in delignifying lignocellulose, thereby enhancing cellulosedegradation. Recently, it has been reported that cellobiosedehydrogenases can enhance the activity of cellulolytic enhancingproteins from Glycosyl Hydrolase Family 61 (See e.g., WO2010/080532A1),and may find use in reactions for redox balance purposes.

Contrary to general understanding in the art, the present inventionprovides genetic modifications (e.g., deletion) of a cellobiosedehydrogenase-encoding gene in cellulase-producing fungal cells. Thismodification results in an improvement in the yield of fermentablesugars from enzyme mixtures secreted by the genetically modified cells.Thus, reduction of cellobiose dehydrogenase secreted by acellulase-producing organism provides a mixture of cellulase enzymesthat can improve yield of fermentable sugars during enzymatic hydrolysisof cellulose-containing substrates. In addition, deletion of the cdhgene provides additional room in the fungal cell genome for introductionof other sequences (e.g., heterologous sequences encoding proteins ofinterest).

Accordingly, provided herein is a fungal cell that has been geneticallymodified to reduce the amount of endogenous cellobiose dehydrogenaseactivity that is secreted by the cell, wherein the fungal cell is fromthe family Chaetomiaceae, and wherein the fungal cell is capable ofsecreting a cellulose-containing enzyme mixture. In some embodiments,the fungal cell is capable of secreting an enzyme mixture comprising twoor more cellulase enzymes. In some embodiments, the fungal cell is aChaetomiaceae family member of the genus Achaetomium, Aporothielavia,Chaetomidium, Chaetomium, Corylomyces, Corynascus, Farrowia, Thielavia,Zopfiella, or Myceliophthora. In some embodiments, the geneticallymodified fungal cell provided herein is a Chaetomiaceae family memberselected from the genera Myceliophthora, Thielavia, Corynascus, orChaetomium.

It is recognized that fungal taxonomy continues to undergoreorganization. Thus, it is intended that all aspects of the presentinvention encompass genera and species that have been reclassified,including but not limited to such organisms as Myceliophthorathermophila, which has also been given various other names (e.g.,Sporotrichum thermophile, Sporotrichum thermophilum, Thelaviaheterothallica, Corynascus heterothallica, Chrysoporium thermophilum,and Myceliophthora indica). Indeed, it is intended that the presentinvention encompass all teleomorphs, anamorphs, and synonyms, basionyms,or taxonomic equivalents thereof.

In some embodiments, the fungal cell has been genetically modified toreduce the amount of endogenous cellobiose dehydrogenase activity thatis secreted by the cell. In some embodiments, the fungal cell is aspecies of Myceliophthora, Thielavia, Sporotrichum, Corynascus,Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, orThermoascus. In some embodiments, the fungal cell is Sporotrichumcellulophilum, Thielavia terrestris, Corynascus heterothallicus,Thielavia heterothallica, Chaetomium globosum, Talaromyces stipitatus,or Myceliophthora thermophila. In some embodiments, the fungal cell isan isolated fungal cell.

In some embodiments, the fungal cell has been genetically modified toreduce the amount of endogenous cellobiose dehydrogenase secreted by thecell. In some embodiments, the fungal cell has been genetically modifiedto disrupt the secretion signal peptide of cellobiose dehydrogenase. Insome embodiments, the fungal cell has been genetically modified toreduce the amount of the endogenous cellobiose dehydrogenase expressedby the cell. For example, in some embodiments, the fungal cell isgenetically modified to disrupt a translation initiation sequence, whilein some other embodiments, the fungal cell is genetically modified tointroduce a frameshift mutation in the transcript encoding theendogenous cellobiose dehydrogenase. In some other embodiments, thefungal cell has been genetically modified to reduce the transcriptionlevel of a gene encoding the endogenous cellobiose dehydrogenase. Forexample, in some embodiments, the fungal cell is genetically modified todisrupt the promoter of a gene encoding the endogenous cellobiosedehydrogenase. For example, in some embodiments, the fungal cell isgenetically modified to disrupt the gene encoding the endogenouscellobiose dehydrogenase through use of stop codons, terminatorelimination, transposons, etc. In some additional embodiments, thefungal cell has been genetically modified to at least partially delete agene encoding the endogenous cellobiose dehydrogenase. In some otherembodiments, the fungal cell has been genetically modified to reduce thecatalytic efficiency of the endogenous cellobiose dehydrogenase. In someembodiments, the fungal cell has been genetically modified, such thatone or more residues in an active site of the cellobiose dehydrogenasehave been mutated. In some embodiments, one or more residues in a hemebinding domain of the cellobiose dehydrogenase of the fungal cell havebeen genetically modified. Indeed, it is intended that any suitablemeans for modifying the fungal cell to reduce the amount of cellobiosedehydrogenase expressed and/or secreted by the cell will find use in thepresent invention.

In some embodiments, the cellobiose dehydrogenase is encompassed withinEC 1.1.99.18. In some embodiments, the cellobiose dehydrogenasecomprises an amino acid sequence that is at least about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 100% identical to SEQ ID NO:2.

In some embodiments, the fungal cell further comprises at least one geneencoding at least one cellulose degrading enzyme that is heterologous tothe fungal cell. For example, in some embodiments, the fungal celloverexpresses a homologous or heterologous gene encoding a cellulosedegrading enzyme such as beta-glucosidase. In some embodiments, thefungal cell overexpresses beta-glucosidase and has been geneticallymodified to reduce the amount of endogenous cellobiose dehydrogenaseactivity secreted by the cell.

The present invention also provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, wherein at least one of the two ormore cellulose hydrolyzing enzymes is expressed by a fungal cell asprovided herein. For example, in some embodiments, the fungal cell is acell that has been genetically modified to reduce the amount ofendogenous cellobiose dehydrogenase activity secreted by the cell,wherein the fungal cell is a member of the genus Myceliophthora,Thielavia, Sporotrichum, Corynascus, Acremonium, Chaetomium, Ctenomyces,Scytalidium, Talaromyces, or Thermoascus. In some embodiments, theenzyme mixture is a cell-free mixture. In some additional embodiments, asubstrate of the enzyme mixture comprises pretreated lignocellulose. Insome embodiments, the pretreated lignocellulose comprises lignocellulosetreated by acid pretreatment, ammonia pretreatment, steam explosion,and/or organic solvent extraction. In some embodiments, the enzymemixture further comprises at least one cellulose degrading enzyme thatis heterologous to the fungal cell. In some embodiments, at least one ofthe two or more cellulose hydrolyzing enzymes is expressed by anisolated fungal cell.

The present invention also provides methods for generating glucose thatcomprise contacting cellulose with a mixture of at least two enzymes.For example, in some embodiments, the methods comprise contactingcellulose with an enzyme mixture comprising two or more cellulosehydrolyzing enzymes, wherein at least one of the two or more cellulosehydrolyzing enzymes is expressed by a fungal cell as described herein.In some embodiments, the methods comprise contacting cellulose with anenzyme mixture comprising two or more cellulose hydrolyzing enzymes,wherein at least one of the two or more cellulose hydrolyzing enzymes isexpressed by a cell that has been genetically modified to reduce theamount of endogenous cellobiose dehydrogenase activity secreted by thecell, wherein the fungal cell is Myceliophthora, Thielavia,Sporotrichum, Corynascus, Acremonium, Chaetomium, Ctenomyces,Scytalidium, Talaromyces, or Thermoascus. In some embodiments, themethods result in an increased yield of glucose and/or cellobiose fromthe hydrolyzed cellulose and decreased oxidation of the cellobiose tooxidized sugar products, such as gluconolactone, gluconate, gluconicacid, cellobionolactone, and/or cellobionic acid from the hydrolyzedcellulose.

In some embodiments, the enzyme mixture is a cell-free mixture. In somefurther embodiments, the cellulose substrate comprises pretreatedlignocellulose. In some additional embodiments, the pretreatedlignocellulose comprises lignocellulose treated by at least onetreatment method such as acid pretreatment, ammonia pretreatment, steamexplosion and/or organic solvent extraction.

In some embodiments, the methods further comprise fermentation of theglucose to an end product such as a fuel alcohol or a precursorindustrial chemical. In some embodiments, the fuel alcohol is ethanol orbutanol. In some embodiments, the methods comprise contacting cellulosewith an enzyme mixture that further comprises a cellulose degradingenzyme that is heterologous to the fungal cell.

Also provided herein are fermentation media comprising the fungal cellof any of the above embodiments, and/or comprising the enzyme mixturederived from the fungal cell of any of the above embodiments.

Definitions

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,protein engineering, and microbiology, which are within the skill of theart. Such techniques are well-known and described in numerous texts andreference works well known to those of skill in the art. All patents,patent applications, articles and publications mentioned herein, bothsupra and infra, are hereby expressly incorporated herein by reference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Many technicaldictionaries are known to those of skill in the art. Although anysuitable methods and materials similar or equivalent to those describedherein find use in the practice of the present invention, some preferredmethods and materials are described herein. It is to be understood thatthis invention is not limited to the particular methodology, protocols,and reagents described, as these may vary, depending upon the contextthey are used by those of skill in the art. Accordingly, the termsdefined immediately below are more fully described by reference to theapplication as a whole.

Also, as used herein, the singular “a”, “an,” and “the” include theplural references, unless the context clearly indicates otherwise.Numeric ranges are inclusive of the numbers defining the range. Thus,every numerical range disclosed herein is intended to encompass everynarrower numerical range that falls within such broader numerical range,as if such narrower numerical ranges were all expressly written herein.It is also intended that every maximum (or minimum) numerical limitationdisclosed herein includes every lower (or higher) numerical limitation,as if such lower (or higher) numerical limitations were expresslywritten herein. Furthermore, the headings provided herein are notlimitations of the various aspects or embodiments of the invention whichcan be had by reference to the application as a whole. Accordingly, theterms defined immediately below are more fully defined by reference tothe application as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

As used herein, “substrate” refers to a substance or compound that isconverted or meant to be converted into another compound by the actionof an enzyme. The term includes not only a single compound but alsocombinations of compounds, such as solutions, mixtures and othermaterials which contain at least one substrate.

As used herein, “conversion” refers to the enzymatic transformation of asubstrate to the corresponding product. “Percent conversion” refers tothe percent of the substrate that is converted to the product within aperiod of time under specified conditions. Thus, for example, the“enzymatic activity” or “activity” of a cellobiose dehydrogenase (“CDH”or “cdh”) polypeptide can be expressed as “percent conversion” of thesubstrate to the product.

As used herein, “secreted activity” refers to enzymatic activity ofcellobiose oxidizing enzymes produced by a fungal cell that is presentin an extracellular environment. An extracellular environment can be,for example, an extracellular milieu such as a culture medium. Thesecreted activity is influenced by the total amount of cellobioseoxidizing enzyme secreted, and also is influenced by the catalyticefficiency of the secreted cellobiose oxidizing enzyme.

As used herein, a “reduction in catalytic efficiency” refers to areduction in the activity of the cellobiose oxidizing enzyme, relativeto unmodified cellobiose oxidizing enzyme, as measured using standardtechniques, as provided herein or otherwise known in the art.

As used herein, the term “enzyme mixture” refers to a combination of atleast two enzymes. In some embodiments, at least two enzymes are presentin a composition. In some additional embodiments, the enzyme mixturesare present within a cell (e.g., a fungal cell). In some embodiments,each or some of the enzymes present in an enzyme mixture are produced bydifferent fungal cells and/or different fungal cultures. In some furtherembodiments, all of the enzymes present in an enzyme mixture areproduced by the same cell. In some embodiments, the enzyme mixturescomprise cellulase enzymes, while in some additional embodiments, theenzyme mixtures comprise enzymes other than cellulases. In someembodiments, the enzyme mixtures comprise at least one cellulase and atleast one enzyme other than a cellulase. In some embodiments, the enzymemixtures comprise enzymes including, but not limited to endoxylanases(EC 3.2.1.8), beta-xylosidases (EC 3.2.1.37),alpha-L-arabinofuranosidases (EC 3.2.1.55), alpha-glucuronidases (EC3.2.1.139), acetylxylanesterases (EC 3.1.1.72), feruloyl esterases (EC3.1.1.73), coumaroyl esterases (EC 3.1.1.73), alpha-galactosidases (EC3.2.1.22), beta-galactosidases (EC 3.2.1.23), beta-mannanases (EC3.2.1.78), beta-mannosidases (EC 3.2.1.25), endo-polygalacturonases (EC3.2.1.15), pectin methyl esterases (EC 3.1.1.11), endo-galactanases (EC3.2.1.89), pectin acetyl esterases (EC 3.1.1.6), endo-pectin lyases (EC4.2.2.10), pectate lyases (EC 4.2.2.2), alpha rhamnosidases (EC3.2.1.40), exo-galacturonases (EC 3.2.1.82), exo-galacturonases (EC3.2.1.67), exopolygalacturonate lyases (EC 4.2.2.9), rhamnogalacturonanendolyases EC (4.2.2.B3), rhamnogalacturonan acetylesterases (EC3.2.1.B11), rhamnogalacturonan galacturonohydrolases (EC 3.2.1.B11),endo-arabinanases (EC 3.2.1.99), laccases (EC 1.10.3.2),manganese-dependent peroxidases (EC 1.10.3.2), amylases (EC 3.2.1.1),glucoamylases (EC 3.2.1.3), lipases, lignin peroxidases (EC 1.11.1.14),and/or proteases.

In some additional embodiments, the present invention further providesenzyme mixtures comprising at least one expansin and/or expansin-likeprotein, such as a swollenin (See e.g., Salheimo et al., Eur. J.Biochem., 269:4202-4211 [2002]) and/or a swollenin-like protein.Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Insome embodiments, an expansin-like protein and/or swollenin-like proteincomprises one or both of such domains and/or disrupts the structure ofcell walls (e.g., disrupting cellulose structure), optionally withoutproducing detectable amounts of reducing sugars. In some additionalembodiments, the enzyme mixtures comprise at least one polypeptideproduct of a cellulose integrating protein, scaffoldin and/or ascaffoldin-like protein (e.g., CipA or CipC from Clostridiumthermocellum or Clostridium cellulolyticum respectively). In someadditional embodiments, the enzyme mixtures comprise at least onecellulose induced protein and/or modulating protein (e.g., as encoded bycipl or cip2 gene and/or similar genes from Trichoderma reesei; Seee.g., Foreman et al., J. Biol. Chem., 278:31988-31997 [2003]). In someadditional embodiments, the enzyme mixtures comprise at least one memberof each of the classes of the polypeptides described above, severalmembers of one polypeptide class, or any combination of thesepolypeptide classes to provide enzyme mixtures suitable for varioususes. Any combination of at least one two, three, four, five, or morethan five enzymes and/or polypeptides find use in various enzymemixtures provided herein. Indeed, it is not intended that the enzymemixtures of the present invention be limited to any particular enzymes,polypeptides, nor combinations, as any suitable enzyme mixture finds usein the present invention.

As used herein, the term “saccharide” refers to any carbohydratecomprising monosaccharides (e.g., glucose, ribose, fructose, galactose,etc.), disaccharides (e.g., sucrose, lactose, maltose, cellobiose,trehalose, melibiose, etc.), oligosaccharides (e.g., raffinose,stachyose, amylose, etc.), and polysaccharides (e.g., starch, glycogen,cellulose, chitin, xylan, arabinoxylan, mannan, fucoidan, galactomannan,callose, laminarin, chrysolaminarin, amylopectin, dextran, dextrins,maltodextrins, inulin, oligofructose, polydextrose, etc.). The termencompasses simple carbohydrates, as well as complex carbohydrates.Indeed, it is not intended that the present invention be limited to anyparticular saccharide, as various saccharides and forms of saccharidesfind use in the present invention.

As used herein, the term “saccharide hydrolyzing enzyme” refers to anyenzyme that hydrolyzes at least one sachharide.

As used herein, the terms “cellobiose oxidizing enzyme” refer to enzymesthat oxidize cellobiose. In some embodiments, cellobiose oxidizingenzymes include cellobiose dehydrogenase (EC 1.1.99.18).

As used herein, the terms “cellobiose dehydrogenase,” “CDH,” and “cdh”refer to a cellobiose: acceptor 1-oxidoreductase that catalyzes theconversion of cellobiose in the presence of an acceptor tocellobiono-1,5-lactone and a reduced acceptor. Examples of cellobiosedehydrogenases fall into the enzyme classification (E.C. 1.1.99.18).Typically 2,6-dichloroindophenol can act as acceptor, as can iron,especially Fe(SCN)₃, molecular oxygen, ubiquinone, or cytochrome C, andother polyphenolics, such as lignin. Substrates of the enzyme includecellobiose, cello-oligosaccharides, lactose, andD-glucosyl-1,4-β-D-mannose, glucose, maltose, mannobiose,thiocellobiose, galactosyl-mannose, xylobiose, xylose. Electron donorsinclude beta-1-4 dihexoses with glucose or mannose at the reducing end,though alpha-1-4 hexosides, hexoses, pentoses, and beta-1-4 pentomerscan act as substrates for at least some of these enzymes (See e.g.,Henriksson et al, Biochim. Biophys. Acta—Prot. Struct. Mol. Enzymol.,1383: 48-54 [1998]; and Schou et al., Biochem. J., 330: 565-571 [1998]).In some embodiments, the cellobiose dehydrogenase of interest in thepresent invention is CDH1, which is encoded by the cdh1 gene.

As used herein, the terms “oxidation”, “oxidize(d)” and the like as usedherein refer to the enzymatic formation of one or more cellobioseoxidation products. When used in reference to a percentage of oxidizedcellobiose, those percentages reflect a weight percent (w/w) relative tothe initial amount of substrate. For example, when the enzyme mixture iscontacted with cellobiose, the percentage of oxidized cellobiosereflects a weight percent (w/w) relative to the initial amount ofcellobiose present in solution. Where the enzyme mixture is contactedwith a cellulose substrate, the percentage of oxidized cellobiosereflects a weight percent (w/w) based on the maximum amount (wt %) ofglucose that could be produced from the total hydrolyzed cellulose(i.e., Gmax).

As used herein, “cellulose” refers to a polymer of the simple sugarglucose linked by beta-1,4 glycosidic bonds.

As used herein, “cellobiose” refers to a water-soluble beta-1,4-linkeddimer of glucose.

As used herein, the term “cellodextrin” refers to a gluocose polymer ofvarying length (i.e., comprising at least two glucose monomers). Eachglucose monomer is linked via a beta-1,4 glycosidic bond. A cellodextrinis classified by its degree of polymerization (DP), which indicates thenumber of glucose monomers the cellodextrin contains. The most commoncellodextrins are: cellobiose (DP=2); cellotriose (DP=3); cellotetrose(DP=4); cellopentose (DP=5); and cellohexose (DP=6). In someembodiments, cellodextrins have a DP of 2-6 (i.e., cellobiose,cellotriose, cellotetrose, cellopentose, and/or cellohexose). In someembodiments, cellodextrins have a DP greater than 6. The degree ofpolymerization of cellodextin molecules can be measured (e.g., by massspectrometry, including but not limited to matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry and electrosprayionization ion trap (ESI-IT) mass spectrometry). Methods of measuringthe degree of polymerization of cellodextrin molecules are known in theart (See e.g., Melander et al., Biomacromol., 7:1410-1421 [2006]).

As used herein, the term “cellulase” refers to any enzyme that iscapable of degrading cellulose. Thus, the term encompasses enzymescapable of hydrolyzing cellulose (β-1,4-glucan or β-D-glucosidiclinkages) to shorter cellulose chains, oligosaccharides, cellobioseand/or glucose. “Cellulases” are divided into three sub-categories ofenzymes: 1,4-β-D-glucan glucanohydrolase (“endoglucanase” or “EG”);1,4-β-D-glucan cellobiohydrolase (“exoglucanase,” “cellobiohydrolase,”or “CBH”); and β-D-glucoside-glucohydrolase (“β-glucosidase,”“cellobiase,” “BG,” or “BGL”). These enzymes act in concert to catalyzethe hydrolysis of cellulose-containing substrates. Endoglucanases breakinternal bonds and disrupt the crystalline structure of cellulose,exposing individual cellulose polysaccharide chains (“glucans”).Cellobiohydrolases incrementally shorten the glucan molecules, releasingmainly cellobiose units (a water-soluble β-1,4-linked dimer of glucose)as well as glucose, cellotriose, and cellotetrose. Beta-glucosidasessplit the cellobiose into monomers. Cellulases often comprise a mixtureof different types of cellulolytic enzymes (endoglucanases andcellobiohydrolases) that act synergistically to break down the celluloseto soluble di- or oligosaccharides such as cellobiose, which are thenfurther hydrolyzed to glucose by beta-glucosidase. Cellulase enzymes areproduced by a wide variety of microorganisms. Cellulases (andhemicellulases) from filamentous fungi and some bacteria are widelyexploited for many industrial applications that involve processing ofnatural fibers to sugars.

As used herein, a “cellulase-producing fungal cell” is a fungal cellthat produces at least one cellulase enzyme (i.e., “cellulosehydrolyzing enzyme”). In some embodiments, the cellulase-producingfungal cells provided herein express and secrete a mixture of cellulosehydrolyzing enzymes.

As used herein, the terms “cellulose hydrolyzing enzyme,” “cellulolyticenzyme,” and like terms refer to an enzyme that acts in the process ofbreaking down cellulose to soluble di- or oligosaccharides such ascellobiose, which are then further hydrolyzed to glucose bybeta-glucosidase. A mixture of cellulose hydrolyzing enzymes is alsoreferred to herein as “cellulases,” a “cellulase-containing mixture,”and/or a “cellulase mixture.”

As used herein, the terms “endoglucanase” and “EG” refer to a categoryof cellulases (EC 3.2.1.4) that catalyze the hydrolysis of internalβ-1,4 glucosidic bonds of cellulose. The term “endoglucanase” is furtherdefined herein as an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase(E.C. 3.2.1.4), which catalyses endohydrolysis of 1,4-beta-D-glycosidiclinkages in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethyl cellulose), lichenan, beta-1,4 bonds in mixedbeta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and otherplant material containing cellulosic components. Endoglucanase activitycan be determined based on a reduction in substrate viscosity orincrease in reducing ends determined by a reducing sugar assay (Seee.g., Zhang et al., Biotechnol. Adv., 24:452-481 [2006]). For purposesof the present invention, endoglucanase activity is determined usingcarboxymethyl cellulose (CMC) hydrolysis (See e.g., Ghose, Pur. Appl.Chem., 59:257-268 [1987]).

As used herein, “EG1” refers to a carbohydrate active enzyme expressedfrom a nucleic acid sequence coding for a glycohydrolase (GH) Family 7catalytic domain classified under EC 3.2.1.4 or any protein, polypeptideor catalytically active fragment thereof. In some embodiments, the EG1is functionally linked to a carbohydrate binding module (CBM), such as aFamily 1 cellulose binding domain

As used herein, the term “EG2” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 5 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or catalytically active fragment thereof. In someembodiments, the EG2 is functionally linked to a carbohydrate bindingmodule (CBM), such as a Family 1 cellulose binding domain.

As used herein, the term “EG3” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 12 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or catalytically active fragment thereof. In someembodiments, the EG3 is functionally linked to a carbohydrate bindingmodule (CBM), such as a Family 1 cellulose binding domain.

As used herein, the term “EG4” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 61 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or fragment thereof. In some embodiments, the EG4 isfunctionally linked to a carbohydrate binding module (CBM), such as aFamily 1 cellulose binding domain

As used herein, the term “EG5” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 45 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or fragment thereof. In some embodiments, the EG5 isfunctionally linked to a carbohydrate binding module (CBM), such as aFamily 1 cellulose binding domain

As used herein, the term “EG6” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 6 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or fragment thereof. In some embodiments, the EG6 isfunctionally linked to a carbohydrate binding module (CBM), such as aFamily 1 cellulose binding domain

As used herein, the terms “cellobiohydrolase” and “CBH” refer to acategory of cellulases (EC 3.2.1.91) that hydrolyze glycosidic bonds incellulose. The term “cellobiohydrolase” is further defined herein as a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (See e.g., Teeri, Tr. Biotechnol., 15:160-167 [1997]; andTeeri et al., Biochem. Soc. Trans., 26:173-178 [1998]). In someembodiments, cellobiohydrolase activity is determined using afluorescent disaccharide derivative4-methylumbelliferyl-.beta.-D-lactoside (See e.g., van Tilbeurgh et al.,FEBS Lett., 149:152-156 [1982]; and van Tilbeurgh and Claeyssens, FEBSLett., 187:283-288 [1985]).

As used herein, the terms “CBH1” and “type 1 cellobiohydrolase” refer toa carbohydrate active enzyme expressed from a nucleic acid sequencecoding for a glycohydrolase (GH) Family 7 catalytic domain classifiedunder EC 3.2.1.91 or any protein, polypeptide or catalytically activefragment thereof. In some embodiments, the CBH1 is functionally linkedto a carbohydrate binding module (CBM), such as a Family 1 cellulosebinding domain.

As used herein, the terms “CBH2” and “type 2 cellobiohydrolase” refer toa carbohydrate active enzyme expressed from a nucleic sequence codingfor a glycohydrolase (GH) Family 6 catalytic domain classified under EC3.2.1.91 or any protein, polypeptide or catalytically active fragmentthereof. Type 2 cellobiohydrolases are also commonly referred to as “theCe16 family.” In some embodiments, the CBH2 is functionally linked to acarbohydrate binding module (CBM), such as a Family 1 cellulose bindingdomain

As used herein, the terms “beta-glucosidase,” “cellobiase,” and “BGL”refers to a category of cellulases (EC 3.2.1.21) that catalyze thehydrolysis of cellobiose to glucose. The term “beta-glucosidase” isfurther defined herein as a beta-D-glucoside glucohydrolase (E.C.3.2.1.21), which catalyzes the hydrolysis of terminal non-reducingbeta-D-glucose residues with the release of beta-D-glucose.Beta-glucosidase activity can be determined using any suitable method(See e.g., J. Basic Microbiol., 42: 55-66 [2002]). One unit ofbeta-glucosidase activity is defined as 1.0 pmole of p-nitrophenolproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

As used herein, the term “glycoside hydrolase 61” and “GH61” refers to acategory of cellulases that enhance cellulose hydrolysis when used inconjunction with one or more additional cellulases. The GH61 family ofcellulases is described, for example, in the Carbohydrate Active Enzymes(CAZY) database (See e.g., Harris et al., Biochem., 49(15):3305-16[2010]).

A “hemicellulase” as used herein, refers to a polypeptide that cancatalyze hydrolysis of hemicellulose into small polysaccharides such asoligosaccharides, or monomeric saccharides. Hemicellulloses includexylan, glucuonoxylan, arabinoxylan, glucomannan and xyloglucan.Hemicellulases include, for example, the following: endoxylanases,beta-xylosidases, alpha-L-arabinofuranosidases, alpha -D-glucuronidases,feruloyl esterases, coumaroyl esterases, alpha-galactosidases,beta-galactosidases, beta-mannanases, and beta-mannosidases.

As used herein, the terms “xylan degrading activity” and “xylanolyticactivity” are defined herein as a biological activity that hydrolyzesxylan-containing material. The two basic approaches for measuringxylanolytic activity include: (1) measuring the total xylanolyticactivity, and (2) measuring the individual xylanolytic activities(endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases) (See e.g., Biely and Puchard, J. Sci. FoodAgr. 86:1636-1647 [2006]; Spanikova and Biely, FEBS Lett., 580:4597-4601[2006]; and Herrmann et al., Biochem. J., 321:375-381 [1997]).

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including oat spelt,beechwood, and larchwood xylans, or by photometric determination of dyedxylan fragments released from various covalently dyed xylans. A commontotal xylanolytic activity assay is based on production of reducingsugars from polymeric 4-O-methyl glucuronoxylan (See e.g., Bailey etal., J. Biotechnol., 23:257-270 [1992]). In some embodiments, xylandegrading activity is determined by measuring the increase in hydrolysisof birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) byxylan-degrading enzyme(s) under the following typical conditions: 1 mLreactions, 5 mg/mL substrate (total solids), 5 mg of xylanolyticprotein/g of substrate, 50 mM sodium acetate pH 5, 50° C., 24 hours,sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay (Seee.g., Lever, Anal. Biochem., 47:273-279 [1972]).

As used herein the term “xylanase activity” refers to a1,4-beta-D-xylan-xylohydrolase activity (E.C. 3.2.1.8) that catalyzesthe endo-hydrolysis of 1,4-beta-D-xylosidic linkages in xylans. In someembodiments, xylanase activity is determined using birchwood xylan assubstrate. One unit of xylanase activity is defined as 1.0 μmole ofreducing sugar (measured in glucose equivalents; See e.g., Lever, Anal.Biochem., 47:273-279 [1972]) produced per minute during the initialperiod of hydrolysis at 50° C., pH 5 from 2 g of birchwood xylan perliter as substrate in 50 mM sodium acetate containing 0.01% TWEEN® 20.

As used herein, the term “beta-xylosidase activity” refers to abeta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes theexo-hydrolysis of short beta (1→4)-xylooligosaccharides, to removesuccessive D-xylose residues from the non-reducing termini In someembodiments of the present invention, one unit of beta-xylosidaseactivity is defined as 1.0 μmole of p-nitrophenol produced per minute at40° C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100mM sodium citrate containing 0.01% TWEEN® 20.

As used herein, the term “acetylxylan esterase activity” refers to acarboxylesterase activity (EC 3.1.1.72) that catalyses the hydrolysis ofacetyl groups from polymeric xylan, acetylated xylose, acetylatedglucose, alpha-napthyl acetate, and p-nitrophenyl acetate. In someembodiments of the present invention, acetylxylan esterase activity isdetermined using 0.5 mM p-nitrophenylacetate as substrate in 50 mMsodium acetate pH 5.0 containing 0.01% TWEEN® 20. One unit ofacetylxylan esterase activity is defined as the amount of enzyme capableof releasing 1 pmole of p-nitrophenolate anion per minute at pH 5, 25°C.

As used herein, the term “feruloyl esterase activity” refers to a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase activity (EC 3.1.1.73) thatcatalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. In some embodiments of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase activityequals the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

As used herein, the term “alpha-glucuronidase activity” refers to analpha-D-glucosiduronate glucuronohydrolase activity (EC 3.2.1.139) thatcatalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronateand an alcohol. One unit of alpha-glucuronidase activity equals theamount of enzyme capable of releasing 1 pmole of glucuronic or4-O-methylglucuronic acid per minute at pH 5, 40° C. (See e.g., deVries, J. Bacteriol., 180:243-249 [1998]).

As used herein the term “alpha-L-arabinofuranosidase activity” refers toan alpha-L-arabinofuranoside arabinofuranohydrolase activity (EC3.2.1.55) that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeactivity acts on alpha-L-arabinofuranosides, alpha-L-arabinanscontaining (1,3)-and/or (1,5)-linkages, arabinoxylans, andarabinogalactans. Alpha-L-arabinofuranosidase is also known asarabinosidase, alpha-arabinosidase, alpha-L-arabinosidase,alpha-arabinofuranosidase, arabinofuranosidase, polysaccharidealpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,L-arabinosidase and alpha-L-arabinanase. For purposes of the presentinvention, alpha-L-arabinofuranosidase activity is determined using 5 mgof medium viscosity wheat arabinoxylan (Megazyme International Ireland,Ltd., Bray, Co. Wicklow, Ireland) per mL of 100 mM sodium acetate pH 5in a total volume of 200 μL for 30 minutes at 40° C. followed byarabinose analysis by AMINEX®. HPX-87H column chromatography (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Enzymatic lignin depolymerization can be accomplished by ligninperoxidases, manganese peroxidases, laccases and cellobiosedehydrogenases (CDH), often working in synergy. These extracellularenzymes, essential for lignin degradation, are often referred to as“lignin-modifying enzymes” or “LMEs.” Three of these enzymes comprisetwo glycosylated heme-containing peroxidases: lignin peroxidase (LIP);Mn-dependent peroxidase (MNP); and, a copper-containing phenoloxidaselaccase (LCC). Although the details of the reaction scheme of ligninbiodegradation are not fully understood to date, without being bound bytheory, it is suggested that these enzymes employ free radicals fordepolymerization reactions.

As used herein, the term “laccase” refers to the copper containingoxidase enzymes that are found in many plants, fungi and microorganisms.Laccases are enzymatically active on phenols and similar molecules andperform a one electron oxidation. Laccases can be polymeric and theenzymatically active form can be a dimer or trimer.

As used herein, the term “Mn-dependent peroxidase” refers to peroxidasesthat require Mn. The enzymatic activity of Mn-dependent peroxidase (MnP)in is dependent on Mn²⁺. Without being bound by theory, it has beensuggested that the main role of this enzyme is to oxidize Mn²⁺ to Mn³⁺(See e.g., Glenn et al. Arch. Biochem. Biophys., 251:688-696 [1986]).Subsequently, phenolic substrates are oxidized by the Mn³⁺ generated.

As used herein, the term “lignin peroxidase” refers to an extracellularheme that catalyses the oxidative depolymerization of dilute solutionsof polymeric lignin in vitro. Some of the substrates of LiP, mostnotably 3,4-dimethoxybenzyl alcohol (veratryl alcohol, VA), are activeredox compounds that have been shown to act as redox mediators. VA is asecondary metabolite produced at the same time as LiP by ligninolyticcultures of P. chrysosporium and without being bound by theory, has beenproposed to function as a physiological redox mediator in theLiP-catalysed oxidation of lignin in vivo (See e.g., Harvey et al., FEBSLett. 195:242-246 [1986]).

As used herein, the term “glucoamylase” (EC 3.2.1.3) refers to enzymesthat catalyze the release of D-glucose from non-reducing ends of oligo-and poly-saccharide molecules. Glucoamylase is also generally considereda type of amylase known as amylo-gludosidase.

As used hererin, the term “amylase” (EC 3.2.1.1) refers to starchcleaving enzymes that degrade starch and related compounds byhydrolyzing the alpha-1,4 and/or alpha-1,6 glucosidic linkages in anendo- or an exo-acting fashion. Amylases include alpha-amylases (EC3.2.1.1); beta-amylases (3.2.1.2), amylo-amylases (EC 3.2.1.3),alpha-glucosidases (EC 3.2.1.20), pullulanases (EC 3.2.1.41), andisoamylases (EC 3.2.1.68). In some embodiments, the amylase is analpha-amylase.

As used herein, the term “pectinase” refers to enzymes that catalyze thehydrolysis of pectin into smaller units such as oligosaccharide ormonomeric saccharides. In some embodiments, the enzyme mixtures compriseany pectinase, for example an endo-polygalacturonase, a pectin methylesterase, an endo-galactanase, a pectin acetyl esterase, an endo-pectinlyase, pectate lyase, alpha rhamnosidase, an exo-galacturonase, anexo-polygalacturonate lyase, a rhamnogalacturonan hydrolase, arhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, arhamnogalacturonan galacturonohydrolase and/or a xylogalacturonase.

As used herein, the term “endo-polygalacturonase” (EC 3.2.1.15) refersto enzymes that catalyze the random hydrolysis of1,4-alpha-D-galactosiduronic linkages in pectate and othergalacturonans. This enzyme may also be referred to as “polygalacturonasepectin depolymerase,” “pectinase,” “endopolygalacturonase,” “pectolase,”“pectin hydrolase,” “pectin polygalacturonase,”“poly-alpha-1,4-galacturonide glycanohydrolase,” “endogalacturonase,”“endo-D-galacturonase,” or” poly(1,4-alpha-D-galacturonide)glycanohydrolase.”

As used herein, the term “pectin methyl esterase” (EC 3.1.1.11) refersto enzymes that catalyze the reaction: pectin+n H₂O=n methanol+pectate.The enzyme may also been known as “pectin esterase,” “pectindemethoxylase,” “pectin methoxylase,” “pectin methylesterase,”“pectase,” “pectinoesterase,” or” pectin pectylhydrolase.”

As used herein, the term “endo-galactanase” (EC 3.2.1.89) refers toenzymes that catalyze the endohydrolysis of 1,4-beta-D-galactosidiclinkages in arabinogalactans. The enzyme may also be known as“arabinogalactan endo-1,4-beta-galactosidase,”“endo-1,4-beta-galactanase,” galactanase,” “arabinogalactanase,” or“arabinogalactan 4-β-D-galactanohydrolase.”

As used herein, the term “pectin acetyl esterase” refers to enzymes thatcatalyze the deacetylation of the acetyl groups at the hydroxyl groupsof GaIUA residues of pectin.

As used herein, the term “one endo-pectin lyase” (EC 4.2.2.10) refers toenzymes that catalyze the eliminative cleavage of(1→4)-alpha-D-galacturonan methyl ester to give oligosaccharides with4-deoxy-6-O-methyl-alpha-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known as “pectin lyase,”“pectin trans-eliminase.” “endo-pectin lyase,” “polymethylgalacturonictranseliminase,” “pectin methyltranseliminase,” “pectolyase,” “PL,”“PNL,” “ PMGL,” or “(1→4)-6-O-methyl-α-D-galacturonan lyase.”

As used herein, the term “pectate lyase” (EC 4.2.2.2) refers to enzymesthat catalyze the eliminative cleavage of (1→4)-alpha-D-galacturonan togive oligosaccharides with 4-deoxy-alpha-D-galact-4-enuronosyl groups attheir non-reducing ends. The enzyme may also be known as“polygalacturonic transeliminase,” “pectic acid transeliminase,”“polygalacturonate lyase,” “endopectin methyltranseliminase,” “pectatetranseliminase,” “endogalacturonate transeliminase,” “pectic acidlyase,” “pectic lyase,” “alpha-1,4-D-endopolygalacturonic acid lyase,”“PGA lyase,” “PPase-N,” “endo-alpha-1,4-polygalacturonic acid lyase,”“polygalacturonic acid lyase,” “pectin trans-eliminase,”“polygalacturonic acid trans-eliminase,” or “(1→4)-alpha-D-galacturonanlyase.”

As used herein, the term “alpha-rhamnosidase” (EC 3.2.1.40) refers toenzymes that catalyze the hydrolysis of terminal non-reducingalpha-L-rhamnose residues in alpha-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known as“alpha-L-rhamnosidase T,” “alpha-L-rhamnosidase N,” or“alpha-L-rhamnoside rhamnohydrolase.”

As used herein, the term “exo-galacturonase” (EC 3.2.1.82) refers toenzymes that hydrolyze pectic acid from the non-reducing end, releasingdigalacturonate. The enzyme may also be known as“exo-poly-alpha-galacturonosidase,” “exopolygalacturonosidase,” or“exopolygalacturanosidase.”

As used herein, the term “exo-galacturan 1,4-alpha galacturonidase” (EC3.2.1.67) refers to enzymes that catalyze reactions of the followingtypes:(1,4-alpha-D-galacturonide)n+H2O=(1,4-alpha-D-galacturonide)n-i+D-galacturonate.The enzyme may also be known as “poly [1->4) alpha-D-galacturonide]galacturonohydrolase,” “exopolygalacturonase,” “poly(galacturonate)hydrolase,” “exo-D-galacturonase,” “exo-D-galacturonanase,”“exopoly-D-galacturonase,” or “poly(1,4-alpha-D-galacturonide)galacturonohydrolase.”

As used herein, the term “exopolygalacturonate lyase” (EC 4.2.2.9)refers to enzymes that catalyze eliminative cleavage of4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducingend of pectate (i.e. de-esterified pectin). This enzyme may be known as“pectate disaccharide-lyase,” “pectate exo-lyase,” “exopectic acidtranseliminase,” “exopectate lyase,” “exopolygalacturonicacid-trans-eliminase,” “PATE,” “exo-PATE,” “exo-PGL,” or“(1→4)-alpha-D-galacturonan reducing-end-disaccharide-lyase.”

As used herein, the term “rhamnogalacturonanase” refers to enzymes thathydrolyze the linkage between galactosyluronic acid and rhamnopyranosylin an endo-fashion in strictly alternating rhamnogalacturonanstructures, consisting of the disaccharide[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

As used herein, the term “rhamnogalacturonan lyase” refers to enzymesthat cleave alpha-L-Rhap-(1→4)-alpha-D-GalpA linkages in an endo-fashionin rhamnogalacturonan by beta-elimination.

As used herein, the term “rhamnogalacturonan acetyl esterase” refers toenzymes that catalyze the deacetylation of the backbone of alternatingrhamnose and galacturonic acid residues in rhamnogalacturonan.

As used herein, the term “rhamnogalacturonan galacturonohydrolase”refers to enzymes that hydrolyze galacturonic acid from the non-reducingend of strictly alternating rhamnogalacturonan structures in anexo-fashion. This enzyme may also be known as “xylogalacturonanhydrolase.”

As used herein, the term “endo-arabinanase” (EC 3.2.1.99) refers toenzymes tha catalyze endohydrolysis of 1,5-alpha-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be known as“endo-arabinase,” “arabinan endo-1,5-α-L-arabinosidase,”“endo-1,5-alpha-L-arabinanase,” “endo-alpha-1,5-arabanase,”“endo-arabanase,” or “1,5-alpha-L-arabinan1,5-alpha-L-arabinanohydrolase.”

As used herein, “protease” includes enzymes that hydrolyze peptide bonds(peptidases), as well as enzymes that hydrolyze bonds between peptidesand other moieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4, and are suitable for use in the presentinvention. Some specific types of proteases include but are not limitedto, cysteine proteases including pepsin, papain and serine proteasesincluding chymotrypsins, carboxypeptidases and metalloendopeptidases.

As used herein, “lipase” includes enzymes that hydrolyze lipids, fattyacids, and acylglycerides, including phosphoglycerides, lipoproteins,diacylglycerols, and the like. In plants, lipids are used as structuralcomponents to limit water loss and pathogen infection. These lipidsinclude waxes derived from fatty acids, as well as cutin and suberin.

As used herein, the terms “isolated” and “purified” are used to refer toa molecule (e.g., an isolated nucleic acid, polypeptide [including, butnot limited to enzymes], etc.) or other component that is removed fromat least one other component with which it is naturally associated.Thus, the terms refer to a material that is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). It is intended that the term encompass any suitable methodfor removing at least one component with which the molecule is naturallyassociated. In some embodiments, the terms also encompass cells that areseparated from other cells and/or media components. It is intended thatany suitable separation method finds use in the present invention. Insome embodiments, a material is said to be “purified” when it is presentin a particular composition in a higher or lower concentration thanexists in a naturally-occurring or wild-type organism or in combinationwith components not normally present upon expression from anaturally-occurring or wild-type organism. For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. In some embodiments, such polynucleotides are partof a vector, and/or such polynucleotides or polypeptides are part of acomposition, and still considered to be isolated, in that such vector orcomposition is not part of its natural environment. In some embodiments,a nucleic acid or protein is said to be purified, for example, if itgives rise to essentially one band in an electrophoretic gel or blot.

The term “isolated,” when used in reference to a DNA sequence, refers toa DNA sequence that has been removed from its natural genetic milieu andis thus free of other extraneous or unwanted coding sequences, and is ina form suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions (e.g., promoters and terminators). Theidentification of associated regions will be evident to one of ordinaryskill in the art (See e.g., Dynan and Tijan, Nature 316:774-78 [1985]).The term “an isolated DNA sequence” is alternatively referred to as “acloned DNA sequence.”

The term “isolated,” when used in reference to a protein, refers to aprotein that is found in a condition other than its native environment.In some embodiments, the isolated protein is substantially free of otherproteins, particularly other homologous proteins. An isolated protein ismore than about 10% pure, preferably more than about 20% pure, and evenmore preferably more than about 30% pure, as determined by SDS-PAGE.Further aspects of the invention encompass the protein in a highlypurified form (i.e., more than about 40% pure, more than about 60% pure,more than about 70% pure, more than about 80% pure, more than about 90%pure, more than about 95% pure, more than about 97% pure, more thanabout 98% pure, or even more than about 99% pure), as determined bySDS-PAGE.

By “purification” or “isolation,” when used in reference to thecellobiose dehydrogenase, it is meant that the cellobiose dehydrogenaseis altered from its natural state by virtue of separating the cellobiosedehydrogenase from some or all of the naturally occurring constituentswith which it is associated in nature. This may be accomplished by anysuitable method, including art recognized separation techniques,including but not limited to ion exchange chromatography, affinitychromatography, hydrophobic separation, dialysis, protease treatment,ammonium sulphate precipitation or other protein salt precipitation,centrifugation, size exclusion chromatography, filtration,microfiltration, gel electrophoresis, separation on a gradient or anyother suitable methods, to remove whole cells, cell debris, impurities,extraneous proteins, or enzymes undesired in the final composition. Itis further possible to then add constituents to the cellobiosedehydrogenase-containing composition which provide additional benefits,for example, activating agents, anti-inhibition agents, desirable ions,compounds to control pH, other enzymes, etc.

As used herein, the phrase “substantially pure polypeptide” refers to acomposition in which the polypeptide species is the predominant speciespresent (i.e., on a molar or weight basis, it is more abundant than anyother individual macromolecular species in the composition), and isgenerally a substantially purified composition when the object speciescomprises at least about 50 percent of the macromolecular speciespresent by mole or % weight. Generally, a substantially pure enzymecomposition will comprise about 60% or more, about 70% or more, about80% or more, about 90% or more, about 95% or more, or about 98% or moreof all macromolecular species by mole or % weight present in thecomposition. Solvent species, small molecules (<500 Daltons), andelemental ion species are not considered macromolecular species.

As used herein, the term “purification process” used in reference to anenzyme mixture encompasses any process that physically removes anundesired component of the enzyme mixture. Thus, in some embodiments,purification processes provided herein include purificationmethodologies that physically remove cellobiose oxidizing activity theenzyme mixture or vice versa. It is contemplated that any suitablepurification process known in the art will find use in the presentinvention. Indeed, it is not intended that the present invention belimited to any particular purification process.

As used herein, the term “cell-free enzyme mixture” comprises enzymesthat have been separated from any cells, including the cells thatsecreted the enzymes. Cell-free enzyme mixtures can be prepared by anyof a variety of methodologies that are known in the art, such asfiltration or centrifugation methodologies. In some embodiments, theenzyme mixture can be, for example, partially cell-free, substantiallycell-free, or entirely cell-free.

As used herein, “polynucleotide” refers to a polymer ofdeoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form, and complements thereof.

A polynucleotide is said to “encode” an RNA or a polypeptide if, in itsnative state or when manipulated by methods known to those of skill inthe art, it can be transcribed and/or translated to produce the RNA, thepolypeptide or a fragment thereof. The anti-sense strand of such anucleic acid is also said to encode the sequences. As is known in theart, DNA can be transcribed by an RNA polymerase to produce RNA, but RNAcan be reverse transcribed by reverse transcriptase to produce a DNA.Thus, a DNA molecule can effectively encode an RNA molecule and viceversa.

The terms “protein” and “polypeptide” are used interchangeably herein torefer to a polymer of amino acid residues. In addition, the terms “aminoacid” “polypeptide,” and “peptide” encompass naturally-occurring andsynthetic amino acids, as well as amino acid analogs. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified (e.g., hydroxyproline,y-carboxyglutamate, and O-phosphoserine).

As used herein, “protein of interest” and “polypeptide of interest”refer to a protein/polypeptide that is desired and/or being assessed. Insome embodiments, the protein of interest is expressed intracellularly,while in other embodiments, it is a secreted polypeptide. In someembodiments, the “protein of interest” or “polypeptide of interest”includes the enzymes of the present invention. In some embodiments, theprotein of interest is a secreted polypeptide which is fused to a signalpeptide (i.e., an amino-terminal extension on a protein to be secreted).Nearly all secreted proteins use an amino-terminal protein extensionwhich plays a crucial role in the targeting to and translocation ofprecursor proteins across the membrane. This extension isproteolytically removed by a signal peptidase during or immediatelyfollowing membrane transfer.

As used herein, the term “amino acid analogs” refers to compounds thathave the same basic chemical structure as a naturally occurring aminoacid (i.e., an a-carbon that is bound to a hydrogen, a carboxyl group,an amino group, and an R group, including but not limited to homoserine,norleucine, methionine sulfoxide, and methionine methyl sulfonium). Insome embodiments, these analogs have modified R groups (e.g.,norleucine) and/or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

An amino acid or nucleotide base “position” is denoted by a number thatsequentially identifies each amino acid (or nucleotide base) in thereference sequence based on its position relative to the N-terminus (or5′-end). Due to deletions, insertions, truncations, fusions, and thelike that must be taken into account when determining an optimalalignment, the amino acid residue number in a test sequence determinedby simply counting from the N-terminus will not necessarily be the sameas the number of its corresponding position in the reference sequence.For example, in a case where a test sequence has a deletion relative toan aligned reference sequence, there will be no amino acid in thevariant that corresponds to a position in the reference sequence at thesite of deletion. Where there is an insertion in an aligned testsequence, that insertion will not correspond to a numbered amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

The terms “wild-type sequence” and “naturally-occurring sequence” areused interchangeably herein, to refer to a polypeptide or polynucleotidesequence that is native or naturally occurring in a host cell. In someembodiments, the wild-type sequence refers to a sequence of interestthat is the starting point of a protein engineering project. Thewild-type sequence may encode either a homologous or heterologousprotein. A homologous protein is one the host cell would produce withoutintervention. A heterologous protein is one that the host cell would notproduce but for intervention.

As used herein, “naturally-occurring enzyme” refers to an enzyme havingthe unmodified amino acid sequence identical to that found in nature(i.e., “wild-type”). Naturally occurring enzymes include native enzymes(i.e., those enzymes naturally expressed or found in the particularmicroorganism).

As used herein, the term “reference enzyme” refers to an enzyme to whichanother enzyme of the present invention (e.g., a “test” enzyme) iscompared in order to determine the presence of an improved property inthe other enzyme being evaluated. In some embodiments, a referenceenzyme is a wild-type enzyme. In some embodiments, the reference enzymeis an enzyme to which a test enzyme of the present invention is comparedin order to determine the presence of an improved property in the testenzyme being evaluated, including but not limited to improvedthermoactivity, improved thermostability, and/or improved stability. Insome embodiments, a reference enzyme is a wild-type enzyme.

As used herein, the term “biologically active fragment,” refers to apolypeptide that has an amino-terminal and/or carboxy-terminaldeletion(s) and/or internal deletion(s), but where the remaining aminoacid sequence is identical to the corresponding positions in thesequence to which it is being compared and that retains substantiallyall of the activity of the full-length polypeptide.

As used herein, the term “recombinant” refers to a polynucleotide orpolypeptide that does not naturally occur in a host cell. In someembodiments, recombinant molecules contain two or morenaturally-occurring sequences that are linked together in a way thatdoes not occur naturally. In some embodiments, “recombinant cells”express genes that are not found in identical form within the native(i.e., non-recombinant) form of the cell and/or express native genesthat are otherwise abnormally over-expressed, under-expressed, and/ornot expressed at all due to deliberate human intervention. Recombinantcells contain at least one recombinant polynucleotide or polypeptide. Anucleic acid construct, nucleic acid (e.g., a polynucleotide),polypeptide, or host cell is referred to herein as “recombinant” when itis non-naturally occurring, artificial or engineered. “Recombination,”“recombining” and generating a “recombined” nucleic acid generallyencompass the assembly of at least two nucleic acid fragments. Thepresent invention also provides a recombinant nucleic acid constructcomprising at least one CDH polynucleotide sequence that hybridizesunder stringent hybridization conditions to the complement of apolynucleotide which encodes a polypeptide having the amino acidsequence of SEQ ID NO:2.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well-characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. As used herein, the term “stringenthybridization wash conditions” in the context of nucleic acidhybridization experiments, such as Southern and Northern hybridizations,are sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen, 1993, “Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes,” Part I,Chapter 2 (Elsevier, N.Y.), which is incorporated herein by reference.For polynucleotides of at least 100 nucleotides in length, low to veryhigh stringency conditions are defined as follows: prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for lowstringencies, 35% formamide for medium and medium-high stringencies, or50% formamide for high and very high stringencies, following standardSouthern blotting procedures. For polynucleotides of at least 100nucleotides in length, the carrier material is finally washed threetimes each for 15 minutes using 2×SSC, 0.2% SDS 50° C. (low stringency),at 55° C. (medium stringency), at 60° C. (medium-high stringency), at65° C. (high stringency), or at 70° C. (very high stringency).

Moderately stringent conditions encompass those known in the art anddescribed in various standard texts and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS). An example of moderately stringent conditions involvesovernight incubation at 37° C. in a solution comprising: 20% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/mLdenatured sheared salmon sperm DNA, followed by washing the filters in1×SSC at about 37-50° C. The skilled artisan will recognize how toadjust the temperature, ionic strength, etc. as necessary to accommodatefactors such as probe length and the like.

As used in some embodiments herein, stringent conditions or highstringency conditions utilize: (1) low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0 1% Ficol1/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) 50% formamide,5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicatedsalmon sperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfate at 42°C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

As used herein, the terms “library of mutants” and “library of variants”used in reference to cells, refer to a population of cells which areidentical in most of their genome but include different homologues ofone or more genes. Such libraries can be used, for example, to identifygenes or operons with improved traits. When used in reference topolypeptides or nucleic acids, “library” refers to a set (i.e., aplurality) of heterogeneous polypeptides or nucleic acids. A library iscomposed of “members.” Sequence differences between library members areresponsible for the diversity present in the library. The library maytake the form of a simple mixture of polypeptides or nucleic acids, ormay be in the form of organisms or cells, for example bacteria, viruses,animal or plant cells and the like, transformed with a library ofnucleic acids.

As used herein, the term “starting gene” refers to a gene of interestthat encodes a protein of interest that is to be improved, deleted,mutated, and/or otherwise changed using the present invention.

The term “property” and grammatical equivalents thereof in the contextof a nucleic acid, as used herein, refer to any characteristic orattribute of a nucleic acid that can be selected or detected. Theseproperties include, but are not limited to, a property affecting bindingto a polypeptide, a property conferred on a cell comprising a particularnucleic acid, a property affecting gene transcription (e.g., promoterstrength, promoter recognition, promoter regulation, and/or enhancerfunction), a property affecting RNA processing (e.g., RNA splicing, RNAstability, RNA conformation, and/or post-transcriptional modification),a property affecting translation (e.g., level, regulation, binding ofmRNA to ribosomal proteins, and/or post-translational modification). Forexample, a binding site for a transcription factor, polymerase,regulatory factor, etc., of a nucleic acid may be altered to producedesired characteristics or to identify undesirable characteristics.

The term “property” and grammatical equivalents thereof in the contextof a polypeptide (including proteins), as used herein, refer to anycharacteristic or attribute of a polypeptide that can be selected ordetected. These properties include, but are not limited to oxidativestability, substrate specificity, catalytic activity, thermal stability,alkaline stability, pH activity profile, resistance to proteolyticdegradation, k_(m), k_(cat), k_(cat)/k_(m) ratio, protein folding,inducing an immune response, not inducing an immune response, ability tobind to a ligand, ability to bind to a receptor, ability to be secreted,ability to be displayed on the surface of a cell, ability tooligomerize, ability to signal, ability to stimulate cell proliferation,ability to inhibit cell proliferation, ability to induce apoptosis,ability to be modified by phosphorylation or glycosylation, and/orability to treat disease, etc. Indeed, it is not intended that thepresent invention be limited to any particular property.

As used herein, “similarity” refers to an identical or conservativeamino acid substitution thereof as defined below. Accordingly, a changeto an identical or conservative substitution for the purposes ofsimilarity is viewed as not comprising a change. A deletion of an aminoacid or a non-conservative amino acid substitution is viewed herein ascomprising a change. Calculation of percent similarity is performed inthe same manner as performed for percent identity.

As used herein, “conservative substitution,” as used with respect toamino acids, refers to the substitution of an amino acid with achemically similar amino acid Amino acid substitutions that do notgenerally alter the specific activity are well known in the art and aredescribed in numerous textbooks. The most commonly occurring exchangesare Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val,Ala/Glu, and Asp/Gly, as well as these in reverse. As used herein, aconservative substitute for a residue is another residue in the samegroup.

In some embodiments, conservative amino acid substitution can be asubstitution such as the conservative substitutions shown in Table A.The substitutions shown are based on amino acid physical-chemicalproperties, and as such, are independent of organism. In someembodiments, the conservative amino acid substitution is a substitutionlisted under the heading of exemplary substitutions.

TABLE A Substitutions Original Residue Conservative SubstitutionsExemplary Substitutions Ala (A) val; leu; ile Val Arg (R) lys; gln; asnLys Asn (N) gln; his; lys; arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln(Q) Asn Asn Glu (E) Asp Asp Gly (G) pro; ala Ala His (H) asn; gln; lys;arg Arg Ile (I) leu; val; met; ala; phe Leu Leu (L) ile; val; met; ala;phe Ile Lys (K) arg; gln; asn Arg Met (M) leu; phe; ile Leu Phe (F) leu;val; ile; ala; tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser SerTrp (W) tyr; phe Tyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu;met; phe; ala Leu

As used herein, the terms “numbered with reference to” or “correspondingto,” when used in the context of the numbering of a given amino acid orpolynucleotide sequence, refers to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence.

The following nomenclature may be used to describe substitutions in areference sequence relative to a reference sequence or a variantpolypeptide or nucleic acid sequence: “R-#-V,” where # refers to theposition in the reference sequence, R refers to the amino acid (or base)at that position in the reference sequence, and V refers to the aminoacid (or base) at that position in the variant sequence.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions. A substitution set can comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acidsubstitutions.

As used herein, “identity” and “percent identity,” in the context of twoor more polypeptide sequences, refers to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues that are the same (e.g., share at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about88% identity, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity) over a specified region to areference sequence, when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using asequence comparison algorithms or by manual alignment and visualinspection. In some embodiments, the terms “percent identity,” “%identity”, “percent identical,” and “% identical,” are usedinterchangeably herein to refer to the percent amino acid orpolynucleotide sequence identity that is obtained by ClustalW analysis(version W 1.8 available from European Bioinformatics Institute,Cambridge, UK), counting the number of identical matches in thealignment and dividing such number of identical matches by the length ofthe reference sequence, and using the following ClustalW parameters toachieve slow/more accurate pairwise optimal alignments—DNA/Protein GapOpen Penalty:15/10; DNA/Protein Gap Extension Penalty:6.66/0.1; Proteinweight matrix: Gonnet series; DNA weight matrix: Identity.

Two sequences are “aligned” when they are aligned for similarity scoringusing a defined amino acid substitution matrix (e.g., BLOSUM62), gapexistence penalty and gap extension penalty so as to arrive at thehighest score possible for that pair of sequences Amino acidsubstitution matrices and their use in quantifying the similaritybetween two sequences are well known in the art (See, e.g., Dayhoff etal., in Dayhoff [ed.], Atlas of Protein Sequence and Structure,” Vol. 5,Suppl. 3, Natl. Biomed. Res. Round., Washington D.C. [1978]; pp.345-352; and Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919[1992], both of which are incorporated herein by reference). TheBLOSUM62 matrix is often used as a default scoring substitution matrixin sequence alignment protocols such as Gapped BLAST 2.0. The gapexistence penalty is imposed for the introduction of a single amino acidgap in one of the aligned sequences, and the gap extension penalty isimposed for each additional empty amino acid position inserted into analready opened gap. The alignment is defined by the amino acid positionof each sequence at which the alignment begins and ends, and optionallyby the insertion of a gap or multiple gaps in one or both sequences soas to arrive at the highest possible score. While optimal alignment andscoring can be accomplished manually, the process is facilitated by theuse of a computer-implemented alignment algorithm (e.g., gapped BLAST2.0; See, Altschul et al., Nucleic Acids Res., 25:3389-3402 [1997],which is incorporated herein by reference), and made available to thepublic at the National Center for Biotechnology Information Website).Optimal alignments, including multiple alignments can be prepared usingreadily available programs such as PSI-BLAST (See e.g., Altschul et al.,supra).

The present invention also provides a recombinant nucleic acid constructcomprising a CDH polynucleotide sequence that hybridizes under stringenthybridization conditions to the complement of a polynucleotide whichencodes a polypeptide having the amino acid sequence of SEQ ID NO:2. Twonucleic acid or polypeptide sequences that have 100% sequence identityare said to be “identical.” A nucleic acid or polypeptide sequence issaid to have “substantial sequence identity” to a reference sequencewhen the sequences have at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, or greater sequence identity as determined usingthe methods described herein, such as BLAST using standard parameters.

As used herein, a “secretion signal peptide” can be a propeptide, aprepeptide or both. For example, the term “propeptide” refers to aprotein precursor that is cleaved to yield a “mature protein.” Thesignal peptide is cleaved from the pre-protein by a signal peptidaseprior to secretion to result in the “mature” or “secreted” protein. Theterms “prepeptide” ad “pre-protein” refer to a polypeptide synthesizedwith an N-terminal signal peptide that targets it for secretion.Accordingly, a “pre-pro-peptide” is a polypeptide that contains a signalpeptide that targets the polypeptide for secretion and which is cleavedoff to yield a mature polypeptide. Signal peptides are found at theN-terminus of the protein and are typically composed of between 6 to 136basic and hydrophobic amino acids.

As used herein, “transcription” and like terms refer to the conversionof the information encoded in a gene to an RNA transcript. Accordingly,a reduction of the transcription level of a cellobiose oxidizing enzymeis a reduction in the amount of RNA transcript of an RNA coding for acellobiose oxidizing enzyme.

As used herein, the terms “DNA construct” and “transforming DNA” areused interchangeably to refer to DNA used to introduce sequences into ahost cell or organism. The DNA may be generated in vitro by PCR or anyother suitable technique(s) known to those in the art. In someembodiments, the DNA construct comprises a sequence of interest (e.g.,as an “incoming sequence”). In some embodiments, the sequence isoperably linked to additional elements such as control elements (e.g.,promoters, etc.). In some embodiments, the DNA construct furthercomprises at least one selectable marker. In some further embodiments,the DNA construct comprises an incoming sequence flanked by homologyboxes. In some further embodiments, the transforming DNA comprises othernon-homologous sequences, added to the ends (e.g., stuffer sequences orflanks). In some embodiments, the ends of the incoming sequence areclosed such that the transforming DNA forms a closed circle. Thetransforming sequences may be wild-type, mutant or modified. In someembodiments, the DNA construct comprises sequences homologous to thehost cell chromosome. In some other embodiments, the DNA constructcomprises non-homologous sequences. Once the DNA construct is assembledin vitro, it may be used to: 1) insert heterologous sequences into adesired target sequence of a host cell; 2) mutagenize a region of thehost cell chromosome (i.e., replace an endogenous sequence with aheterologous sequence); 3) delete target genes; and/or 4) introduce areplicating plasmid into the host. In some embodiments, the incomingsequence comprises at least one selectable marker. This sequence cancode for one or more proteins of interest. It can have other biologicalfunctions. In many cases the incoming sequence comprises at least oneselectable marker, such as a gene that confers antimicrobial resistance.

As used herein, a “vector” is a polynucleotide construct for introducinga polynucleotide sequence into a cell. In some embodiments, the vectorcomprises a suitable control sequence operably linked to and capable ofeffecting the expression of the polypeptide encoded in thepolynucleotide sequence in a suitable host. An “expression vector” has apromoter sequence operably linked to the polynucleotide sequence (e.g.,transgene) to drive expression in a host cell, and in some embodiments atranscription terminator sequence. In some embodiments, the vectors aredeletion vectors. In some embodiments, vectors comprise polynucleotidesequences that produce small interfering RNA or antisense RNAtranscripts that interfere with the translation of a targetpolynucleotide sequence.

As used herein, a “deletion vector” comprises polynucleotide sequenceshomologous to a polynucleotide sequences 5′ and 3′ to a target sequenceto be deleted from a host genome so as to direct recombination andreplacement of the target sequence with a polynucleotiode between the 5′and 3′ targeting sequences.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell. In general theterm, “expression” refers to conversion of the information encoded in agene to the protein encoded by that gene. Thus, a “reduction of theamount of an expressed cellobiose oxidizing enzyme” is a reduction inthe amount of the cellobiose oxidizing enzyme that is eventuallytranslated by the cell.

As used herein, the term “overexpress” is intended to encompassincreasing the expression of a protein to a level greater than the cellnormally produces. It is intended that the term encompass overexpressionof endogenous, as well as heterologous proteins. In some embodiments,overexpression includes an elevated transcription rate and/or level ofthe gene compared to the endogenous transcription rate and/or level forthat gene. For example, in some embodiments, a heterologous gene isintroduced into a fungal cell to express a gene encoding a heterologousenzyme such as a beta-glucosidase from another organism In some otherembodiments, a heterologous gene is introduced into a fungal cell tooverexpress a gene encoding a homologous enzyme such as abeta-glucosidase.

In some embodiments, the heterologous gene is a gene that has beenmodified to overexpress the gene product. In some embodiments,“overexpression” refers to any state in which a gene is caused to beexpressed at an elevated rate or level as compared to the endogenousexpression rate or level for that gene. In some embodiments,overexpression includes elevated translation rate and/or level of thegene compared to the endogenous translation rate and/or level for thatgene.

As used herein, the term “produces” refers to the production of proteinsand/or other compounds by cells. It is intended that the term encompassany step involved in the production of polypeptides including, but notlimited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, a “cellobiose dehydrogenase that is secreted by a cell”is a cellobiose dehydrogenase produced by the cell in a manner such thatthe cellobiose dehydrogenase is exported across a cell membrane and thensubsequently released into the extracellular milieu, such as intoculture media.

As used herein, a “polynucleotide sequence that has been adapted forexpression” is a polynucleotide sequence that has been inserted into anexpression vector or otherwise modified to contain regulatory elementsnecessary for expression of the polynucleotide in the host cell,positioned in such a manner as to permit expression of thepolynucleotide in the host cell. Such regulatory elements required forexpression include promoter sequences, transcription initiationsequences and, optionally, enhancer sequences. For example, in someembodiments, a polynucleotide sequence is inserted into a plasmidadapted for expression in the fungal host cell.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector. In some embodiments,plasmids form an extrachromosomal self-replicating genetic element insome eukaryotes and/or prokaryotes, while in some other embodiments,plasmids integrate into the host cell chromosome.

As used herein, a “control sequence” includes all components, which arenecessary or advantageous for the expression of a polynucleotide of thepresent disclosure. Each control sequence may be native or foreign tothe polynucleotide of interest. Such control sequences include, but arenot limited to, leaders, polyadenylation sequences, propeptidesequences, promoters, signal peptide sequences, and transcriptionterminators.

As used herein, “operably linked” refers to a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

As used herein, a nucleic acid is “operably linked” when it is placedinto a functional relationship with another nucleic acid sequence. Forexample, DNA encoding a secretory leader (i.e., a signal peptide), isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous, and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

As used herein the term “gene” refers to a polynucleotide (e.g., a DNAsegment), that encodes a polypeptide and includes regions preceding andfollowing the coding regions as well as intervening sequences (introns)between individual coding segments (exons).

As used herein, an “endogenous” or “homologous” gene refers to a gene(including, but not limited to wild-type) that is found in a parentalstrain of a host cell (e.g., fungal or bacterial cell). As used hereinin making comparisons between nucleic acid sequences, “homologous genes”(or “homologue” genes) refers to genes from different, but usuallyrelated species, which correspond to each other and which are identicalor very similar to each other. The term encompasses genes that areseparated by speciation (i.e., the development of new species) (e.g.,orthologous genes), as well as genes that have been separated by geneticduplication (e.g., paralogous genes).

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature.

As used herein, a “heterologous enzyme” refers to an enzyme that isencoded by a “heterologous gene.” However, it is also contemplated thata heterologous gene encodes an endogenous or homologous enzyme, asdescribed herein. In general, the term “heterologous gene” refers to agene that occurs in a form not found in a parental strain of the hostfungal cell (including but not limited to wild-type). Thus, in someembodiments, a heterologous gene is a gene that is derived from aspecies that is different from the species of the fungal cell expressingthe gene and recognized anamorphs, teleomorphs or taxonomic equivalentsof the fungal cell expressing the gene. In some embodiments, aheterologous gene is a modified version of a gene that is endogenous tothe host fungal cell, which endogenous gene has been subjected tomanipulation and then introduced or transformed into the host cell. Forexample, in some embodiments, a heterologous gene has an endogenouscoding sequence, but has modifications to the promoter sequence.Similarly, in some embodiments, a heterologous gene encodes the sameamino acid sequence as an endogenous gene, but has modifications to thecodon usage or to noncoding regions such as introns, or a combinationthereof. For example, in some embodiments, a heterologous gene comprisesmodifications to the coding sequence to encode a non-wild typepolypeptide. In some other embodiments, a heterologous gene has the samepromoter sequence, 5′ and 3′ untranslated regions and coding regions asa parental strain, but be located in another region of the samechromosome, or on an entirely different chromosome as compared to aparental strain of the host cell.

As used herein, the term “introduced” used in the context of inserting anucleic acid sequence into a cell, means transformation, transduction,conjugation, transfection, and/or any other suitable method(s) known inthe art for inserting nucleic acid sequences into host cells. Anysuitable means for the introduction of nucleic acid into host cells finduse in the present invention.

As used herein, the terms “transformed” and “transformation” used inreference to a cell refer to a cell that has a non-native nucleic acidsequence integrated into its genome or has an episomal plasmid that ismaintained through multiple generations. In some embodiments, the terms“transformed” and “stably transformed” refer to a cell that has anon-native (i.e., heterologous) polynucleotide sequence integrated intoits genome or as an episomal plasmid that is maintained for at least twogenerations.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising polynucleotidesequences (e.g., DNA) as provided herein. In some embodiments, the hostcells are prokaryotic or eukaryotic cells that have been transformed ortransfected with vectors constructed using recombinant techniques asknown in the art. Transformed hosts are capable of either replicatingvectors encoding at least one protein of interest and/or expressing thedesired protein of interest. In addition, reference to a cell of aparticular strain refers to a parental cell of the strain as well asprogeny and genetically modified derivatives. Genetically modifiedderivatives of a parental cell include progeny cells that contain amodified genome or episomal plasmids that confer for example, antibioticresistance, improved fermentation, etc. In some embodiments, host cellsare genetically modified to have characteristics that improve proteinsecretion, protein stability or other properties desirable forexpression and/or secretion of a protein. For example, knockout of Alp1function results in a cell that is protease deficient. Knockout of pyr5function results in a cell with a pyrimidine deficient phenotype. Insome embodiments, host cells are modified to delete endogenous cellulaseprotein-encoding sequences or otherwise eliminate expression of one ormore endogenous cellulases. In some embodiments, expression of one ormore endogenous cellulases is inhibited to increase production ofcellulases of interest. Genetic modification can be achieved by anysuitable genetic engineering techniques and/or classical microbiologicaltechniques (e.g., chemical or UV mutagenesis and subsequent selection).Using recombinant technology, nucleic acid molecules can be introduced,deleted, inhibited or modified, in a manner that results in increasedyields of enzyme within the organism or in the culture. For example,knockout of Alp1 function results in a cell that is protease deficient.Knockout of pyr5 function results in a cell with a pyrimidine deficientphenotype. In some genetic engineering approaches, homologousrecombination is used to induce targeted gene modifications byspecifically targeting a gene in vivo to suppress expression of theencoded protein. In an alternative approach, siRNA, antisense, and/orribozyme technology finds use in inhibiting gene expression.

The terms “modified sequence” and “modified genes” are usedinterchangeably herein to refer to a sequence that includes a deletion,insertion, substitution or any other interruption of a naturallyoccurring nucleic acid sequence. In some embodiments, the expressionproduct of the modified sequence is a truncated protein (e.g., if themodification is a deletion or interruption of the sequence). In someembodiments, the truncated protein retains biological activity. In somealternative embodiments, the expression product of the modified sequenceis an elongated protein (e.g., modifications comprising an insertioninto the nucleic acid sequence). In some further embodiments, aninsertion leads to a truncated protein (e.g., when the insertion resultsin the formation of a stop codon). Thus, an insertion may result ineither a truncated protein or an elongated protein as an expressionproduct.

As used herein, the terms “mutant nucleic acid sequence,” “mutantnucleotide sequence,” and “mutant gene” are used interchangeably inreference to a nucleotide sequence that has an alteration in at leastone codon occurring in a host cell's wild-type nucleotide sequence. Theexpression product of the mutant sequence is a protein with an alteredamino acid sequence relative to the wild-type. In some embodiments, theexpression product has an altered functional capacity (e.g., enhancedenzymatic activity).

As used herein, the term “targeted randomization” refers to a processthat produces a plurality of sequences where one or several positionshave been randomized In some embodiments, randomization is complete(i.e., all four nucleotides, A, T, G, and C can occur at a randomizedposition). In some alternative embodiments, randomization of anucleotide is limited to a subset of the four nucleotides. Targetedrandomization can be applied to one or several codons of a sequence,coding for one or several proteins of interest. When expressed, theresulting libraries produce protein populations in which one or moreamino acid positions can contain a mixture of all 20 amino acids or asubset of amino acids, as determined by the randomization scheme of therandomized codon. In some embodiments, the individual members of apopulation resulting from targeted randomization differ in the number ofamino acids, due to targeted or random insertion or deletion of codons.In some further embodiments, synthetic amino acids are included in theprotein populations produced. In some additional embodiments, themajority of members of a population resulting from targetedrandomization show greater sequence homology to the consensus sequencethan the starting gene. In some embodiments, the sequence encodes one ormore proteins of interest. In some alternative embodiments, the proteinshave differing biological functions.

As used herein, “deletion” refers to modification of the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 3 or more amino acids, 4 or more amino acids, 5 or more aminoacids, 6 or more amino acids, 7 or more amino acids, 8 or more aminoacids, 9 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thepolypeptide while retaining enzymatic activity and/or retaining theimproved properties of an engineered cellobiose dehydrogenase enzyme.Deletions may be present in the internal portions and/or terminalportions of the polypeptide. In some embodiments, the deletion comprisesa continuous segment, while in other embodiments, it is discontinuous.

As used herein, a “gene deletion” or “deletion mutation” is a mutationin which at least part part of a sequence of the DNA making up the geneis missing. Thus, a “deletion” in reference to nucleic acids is a lossor replacement of genetic material resulting in a complete or partialdisruption of the sequence of the DNA making up the gene. In someembodiments, complete or near-complete deletion of the gene sequence iscontemplated. However, a deletion mutation need not completely removethe entire gene sequence for the cellobiose oxidizing enzyme in order toreduce the endogenous cellobiose oxidizing enzyme activity secreted bythe fungal cell. For example, a partial deletion that removes one ormore nucleotides encoding an amino acid in a cellobiose oxidizing enzymeactive site, encoding a secretion signal, or encoding another portion ofthe cellobiose oxidizing enzyme that plays a role in endogenouscellobiose oxidizing enzyme activity being secreted by the fungal cell.Any number of nucleotides can be deleted, from a single base to anentire piece of a chromosome. Thus, in some embodiments, the term“deletion” refers to the removal of a gene necessary for encoding aspecific protein (e.g., cdh1). In this case, the strain having thisdeletion can be referred to as a “deletion strain.”

As used herein, “fragment” refers to a polypeptide that has anamino-terminal and/or carboxy-terminal and/or internal deletion, ascompared to a reference polypeptide, but where the remaining amino acidsequence is identical to the corresponding positions in the referencesequence. Fragments can typically have about 80%, about 90%, about 95%,about 98%, or about 99% of the full-length cellobiose dehydrogenasepolypeptide, for example the polypeptide of SEQ ID NO:2. In someinstances, the sequences of the non-naturally occurring and wild-typecellobiose dehydrogenase polypeptides disclosed herein can include aninitiating methionine (M) residue (i.e., M at position 1). However, theskilled artisan will recognize that this initiating methionine residuecan be removed during the course of biological processing of the enzyme,such as in a host cell or in vitro translation system, to generate amature enzyme lacking the initiating methionine residue, but otherwiseretaining the enzyme's properties. Thus, for each of the cellobiosedehydrogenase polypeptides disclosed herein having an amino acidsequence comprising an initiating methionine, the present disclosurealso encompasses the polypeptide with the initiating methionine residuedeleted (i.e., a fragment of the cellobiose dehydrogenase polypeptidelacking a methionine at position 1).

As used herein, a “conditional mutation” is a mutation that haswild-type phenotype under certain environmental conditions and a mutantphenotype under certain other conditions.

As used herein, the term “screening” has its usual meaning in the artand is, in general a multi-step process. In the first step, a mutantnucleic acid or variant polypeptide therefrom is provided. In the secondstep, a property of the mutant nucleic acid or variant polypeptide isdetermined In the third step, the determined property is compared to aproperty of the corresponding precursor nucleic acid, to the property ofthe corresponding naturally occurring polypeptide or to the property ofthe starting material (e.g., the initial sequence) for the generation ofthe mutant nucleic acid. It will be apparent to the skilled artisan thatthe screening procedure for obtaining a nucleic acid or protein with analtered property depends upon the property of the starting material, andthe modification of which the generation of the mutant nucleic acid isintended to facilitate. The skilled artisan will therefore appreciatethat the invention is not limited to any specific property to bescreened for and that the following description of properties listsillustrative examples only. Methods for screening for any particularproperty are generally described in the art. For example, one canmeasure binding, pH optima, specificity, etc., before and aftermutation, wherein a change indicates an alteration. In some embodiments,the screens are performed in a high-throughput manner, includingmultiple samples being screened simultaneously, including, but notlimited to assays utilizing chips, phage display, multiple substratesand/or indicators, and/or any other suitable method known in the art.

As used in some embodiments, screens encompass selection steps in whichvariants of interest are enriched from a population of variants.Examples of these embodiments include the selection of variants thatconfer a growth advantage to the host organism, as well as phage displayor any other method of display, where variants can be captured from apopulation of variants based on their binding or catalytic properties.In some embodiments, a library of variants is exposed to stress (e.g.,exposure to heat, protease, or denaturing conditions). Subsequently,variants that are still intact are identified in a screen or enriched byselection. It is intended that the term encompass any suitable means forselection. Indeed, it is not intended that the present invention belimited to any particular method of screening.

As used herein, a “genetically modified” and/or “genetically engineeredcell” (e.g., a “geneticially engineered fungal cell” and/or a“genetically modified fungal cell”) is a cell whose genetic material hasbeen altered using genetic engineering techniques. A geneticallymodified cell also refers to a derivative of or the progeny of a cellwhose genetic material has been altered using genetic engineeringtechniques. An example of a genetic modification as a result of geneticengineering techniques includes a modification to the genomic DNA;another example of a genetic modification as a result of geneticengineering techniques includes introduction of a stable heterologousnucleic acid into the cell. For example, as provided herein, agenetically modified fungal cell as provided herein is a fungal cellthat whose genetic material has been altered in such a way as to eitherreduce the amount of secreted cellobiose oxidizing enzyme activity, orto reduce the ability of the secreted enzyme to oxidize cellobiose.

In some embodiments, mutant DNA sequences are generated using sitesaturation mutagenesis in at least one codon. In some other embodiments,site saturation mutagenesis is performed for two or more codons. In somefurther embodiments, mutant DNA sequences have more than about 50%, morethan about 55%, more than about 60%, more than about 65%, more thanabout 70%, more than about 75%, more than about 80%, more than about85%, more than about 90%, more than about 95%, or more than about 98%homology with the wild-type sequence. In some alternative embodiments,mutant DNA is generated in vivo using any suitable known mutagenicprocedure including, but not limited to the use of radiation,nitrosoguanidine, etc. The desired DNA sequence is then isolated andused in the methods provided herein.

As used herein, the terms “amplification” and “gene amplification” referto a method by which specific DNA sequences are disproportionatelyreplicated such that the amplified gene becomes present in a higher copynumber than was initially present in the genome. In some embodiments,selection of cells by growth in the presence of a drug (e.g., aninhibitor of an inhibitable enzyme) results in the amplification ofeither the endogenous gene encoding the gene product required for growthin the presence of the drug or by amplification of exogenous (i.e.,input) sequences encoding this gene product, or both. “Amplification” isa special case of nucleic acid replication involving templatespecificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a synthesis initiation pointwhen placed under conditions in which synthesis of a primer extensionproduct which is complementary to a nucleic acid strand is induced(i.e., in the presence of nucleotides and an inducing agent such as DNApolymerase and at a suitable temperature and pH). The primer ispreferably single stranded for maximum efficiency in amplification, butmay alternatively be double stranded. If double stranded, the primer isfirst treated to separate its strands before being used to prepareextension products. In some embodiments, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. As known in the art, the exact lengths of the primers will dependon many factors, including temperature, source of primer and the use ofthe method.

The terms “mutagenic primer” and “mutagenic oligonucleotide” (usedinterchangeably herein) refer to oligonucleotide compositions whichcorrespond to a portion of a template sequence and which are capable ofhybridizing thereto. With respect to mutagenic primers, the primer willnot precisely match the template nucleic acid, the mismatch ormismatches in the primer being used to introduce the desired mutationinto the nucleic acid library.

As used herein, the terms “non-mutagenic primer” and “non-mutagenicoligonucleotide” (used interchangeably herein) are intended to refer tooligonucleotide compositions which will match precisely to a templatenucleic acid. In some embodiments of the invention, only mutagenicprimers are used. In some other embodiments, the primers are designed sothat for at least one region at which a mutagenic primer has beenincluded, there is also non-mutagenic primer included in theoligonucleotide mixture. By adding a mixture of mutagenic primers andnon-mutagenic primers corresponding to at least one of the mutagenicprimers, it is possible to produce a resulting nucleic acid library inwhich a variety of combinatorial mutational patterns are presented. Forexample, if it is desired that some of the members of the mutant nucleicacid library retain their precursor sequence at certain positions whileother members are mutant at such sites, the non-mutagenic primersprovide the ability to obtain a specific level of non-mutant memberswithin the nucleic acid library for a given residue. The methods of theinvention employ mutagenic and non-mutagenic oligonucleotides which aregenerally between about 10-50 bases in length, or more preferably, about15-45 bases in length. However, it may be necessary to use primers thatare either shorter than about 10 bases or longer than about 50 bases toobtain the mutagenesis result desired. With respect to correspondingmutagenic and non-mutagenic primers, it is not necessary that thecorresponding oligonucleotides be of identical length, but only thatthere is overlap in the region corresponding to the mutation to beadded. Primers may be added in a pre-defined ratio according to thepresent invention. For example, if it is desired that the resultinglibrary have a significant level of a certain specific mutation and alesser amount of a different mutation at the same or different site, itis possible to produce the desired biased library by adjusting theamount of primer added. Alternatively, by adding lesser or greateramounts of non-mutagenic primers, it is possible to adjust the frequencywith which the corresponding mutation(s) are produced in the mutantnucleic acid library.

As used herein, the phrase “contiguous mutations” refers to mutationswhich are presented within the same oligonucleotide primer. For example,contiguous mutations may be adjacent or nearby each other, however, theywill be introduced into the resulting mutant template nucleic acids bythe same primer.

As used herein, the phrase “discontiguous mutations” refers to mutationswhich are presented in separate oligonucleotide primers. For example,discontiguous mutations will be introduced into the resulting mutanttemplate nucleic acids by separately prepared oligonucleotide primers.

As used herein, the term “degenerate codon” refers to a codon used torepresent a set of different codons (also referred to as an “ambiguouscodon”). For example, the degenerate codon “NNT” represents a set of 16codons having the base triplet sequence (A, C, T, or G)/(A, C, T, orG)/T.

As used herein, “coding sequence” refers to that portion of apolynucleotide that encodes an amino acid sequence of a protein (e.g., agene).

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (PCR) refers to themethods of U.S. Pat. Nos. 4,683,195 4,683,202, and 4,965,188, herebyincorporated by reference, which include methods for increasing theconcentration of a segment of a target sequence in a mixture of genomicDNA without cloning or purification. This method for amplifying thetarget sequence is well known in the art.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A “restriction site” refers to a nucleotide sequence recognized andcleaved by a given restriction endonuclease and is frequently the sitefor insertion of DNA fragments. In some embodiments of the invention,restriction sites are engineered into the selective marker and into 5′and 3′ ends of the DNA construct.

As used herein, “homologous recombination” means the exchange of DNAfragments between two DNA molecules or paired chromosomes at the site ofidentical or nearly identical nucleotide sequences. In some embodiments,chromosomal integration is homologous recombination.

As used herein, the term “C1” refers to Myceliophthora thermophilia,including the fungal strain described by Garg (See, Garg, Mycopathol.,30: 3-4 [1966]).

As used herein, “Chrysosporium lucknowense” includes the strainsdescribed in U.S. Pat. Nos. 6,015,707, 5,811,381 and 6,573,086; U.S.Pat. Pub. Nos. 2007/0238155, US 2008/0194005, U.S. 2009/0099079;International Pat. Pub. Nos., WO 2008/073914 and WO 98/15633, all ofwhich are incorporated herein by reference, and include, withoutlimitation, Chrysosporium lucknowense Garg 27K, VKM-F 3500 D (AccessionNo. VKM F-3500-D), C1 strain UV13-6 (Accession No. VKM F-3632 D), C1strain NG7C-19 (Accession No. VKM F-3633 D), and C1 strain UV18-25 (VKMF-3631 D), all of which have been deposited at the All-RussianCollection of Microorganisms of Russian Academy of Sciences (VKM),Bakhurhina St. 8, Moscow, Russia, 113184, and any derivatives thereof.Although initially described as Chrysosporium lucknowense, C1 maycurrently be considered a strain of Myceliophthora thermophila. Other C1strains include cells deposited under accession numbers ATCC 44006, CBS(Centraalbureau voor Schimmelcultures) 122188, CBS 251.72, CBS 143.77,CBS 272.77, CBS122190, CBS122189, and VKM F-3500D. Exemplary C1derivatives include modified organisms in which one or more endogenousgenes or sequences have been deleted or modified and/or one or moreheterologous genes or sequences have been introduced. Derivativesinclude, but are not limited to UV18#100f Δalpl, UV18#100f Δpyr5 Δalpl,UV18#100.f Δalpl Δpep4 Δalp2, UV18#100.f Δpyr5 Δalpl Δpep4 Δalp2 andUV18#100.f Δpyr4 Δpyr5 ΔaIp1 Δpep4 Δalp2, as described in WO 2008073914and WO 2010107303, each of which is incorporated herein by reference.

As used herein, the term “culturing” refers to growing a population ofmicrobial cells under suitable conditions in a liquid or solid medium.It is contemplated that the culturing be carried out in any suitableformat, equipment (e.g., shake flasks, fermentation tanks, bioreactors,etc.). It is also intended that the culturing be conducted using anysuitable process methods, including but not limited to batch, fed-batch,and/or continuous culturing. Indeed, it is contemplated that anycombination of suitable methods will find use.

In a “batch process,” all the necessary materials, with the exception ofoxygen for aerobic processes, are placed in a reactor at the start ofthe operation and the fermentation is allowed to proceed untilcompletion, at which point the product is harvested. In someembodiments, batch processes for producing the fungal cells, enzymes,and/or enzyme mixtures of the present invention are carried out in ashake-flask or a bioreactor.

In a “fed-batch process,” the culture is fed continuously orsequentially with one or more media components without the removal ofthe culture fluid.

In a “continuous process,” fresh medium is supplied and culture fluid isremoved continuously at volumetrically equal rates to maintain theculture at a steady growth rate. In reference to continous processes,“steady state” refers to a state in which the concentration of reactantsdoes not vary appreciably, and “quasi-steady state” refers to a state inwhich, subsequent to the initiation of the reaction, the concentrationof reactants fluctuates within a range consistent with normal operationof the continuous hydrolysis process.

As used herein, the term “saccharification” refers to the process inwhich substrates (e.g., cellulosic biomass) are broken down via theaction of cellulases to produce fermentable sugars (e.g. monosaccharidessuch as but not limited to glucose).

As used herein, the term “fermentable sugars” refers to simple sugars(e.g., monosaccharides, disaccharides and short oligosaccharides),including but not limited to glucose, xylose, galactose, arabinose,mannose and sucrose. Indeed, a fermentable sugar is any sugar that amicroorganism can utilize or ferment.

As used herein the term “soluble sugars” refers to water-soluble pentoseand hexose monomers and oligomers of up to about six monomer units. Itis intended that the term encompass any water soluble mono- and/oroligosaccharides.

As used herein, the term “fermentation” is used broadly to refer to theprocess of obtaining energy from the oxidation of organic compounds(e.g., carbohydrates). Indeed, “fermentation” broadly refers to thechemical conversion of a sugar source to an end product through the useof a fermenting organism. In some embodiments, the term encompassescultivation of a microorganism or a culture of microorganisms that usesugars, such as fermentable sugars, as an energy source to obtain adesired product.

As used herein, the term “fermenting organism” refers to any organism,including prokaryotic, as well as eukaryotic organisms (e.g., bacterialorganisms, as well as fungal organisms such as yeast and filamentousfungi), suitable for producing a desired end product. Especiallysuitable fermenting organisms are able to ferment (i.e., convert)sugars, including but not limited to glucose, fructose, maltose, xylose,mannose and/or arabinose, directly or indirectly into at least onedesired end product. In some embodiments, yeast that find use in thepresent invention include, but are not limited to strains of the genusSaccharomyces (e.g., strains of Saccharomyces cerevisiae andSaccharomyces uvarum), strains of the genus Pichia (e.g., Pichiastipitis such as Pichia stipitis CBS 5773 and Pichia pastoris), andstrains of the genus Candida (e.g., Candida utilis, Candidaarabinofermentans, Candida diddensii, Candida sonorensis, Candidashehatae, Candida tropicalis, and Candida boidinii). Other fermentingorganisms include, but are not limited to strains of Zymomonas,Hansenula (e.g., Hansenula polymorpha and Hansenula anomala),Kluyveromyces (e.g., Kluyveromyces fragilis), and Schizosaccharomyces(e.g., Schizosaccharomyces pombe).

As used herein, the term “slurry” refers to an aqueous solution in whichare dispersed one or more solid components, such as a cellulosicsubstrate. Thus, the term “slurry” refers to a suspension of solids in aliquid. In some embodiments, the cellulosic substrate is slurried in aliquid at a concentration that is thick, but can still be pumped. Forexample, in some embodiments, the liquid is water, a recycled processstream, and/or a treated effluent. However, it is not intended that thepresent invention be limited to any particular liquid and/or solid.

The terms “biomass,” and “biomass substrate,” encompass any suitablematerials for use in saccharification reactions. The terms encompass,but are not limited to, materials that comprise cellulose (i.e.,“cellulosic biomass,” “cellulosic feedstock,” and “cellulosicsubstrate”), as well as lignocellulosic biomass. Indeed, the term“biomass” encompasses any living or dead biological material thatcontains a polysaccharide substrate, including but not limited tocellulose, starch, other forms of long-chain carbohydrate polymers, andmixtures of such sources. In some embodiments, it is assembled entirelyor primarily from glucose or xylose, and in some embodiments, optionallyalso contains various other pentose and/or hexose monomers. Biomass canbe derived from plants, animals, or microorganisms, and includes, but isnot limited to agricultural, industrial, and forestry residues,industrial and municipal wastes, and terrestrial and aquatic crops grownfor energy purposes. Examples of biomass substrates include, but are notlimited to, wood, wood pulp, paper pulp, corn fiber, corn grain, corncobs, crop residues such as corn husks, corn stover, grasses, wheat,wheat straw, barley, barley straw, hay, rice, rice straw, switchgrass,waste paper, paper and pulp processing waste, woody or herbaceousplants, fruit or vegetable pulp, distillers grain, grasses, rice hulls,cotton, hemp, flax, sisal, sugar cane bagasse, sorghum, soy,switchgrass, components obtained from milling of grains, trees,branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, and flowers and any suitable mixtures thereof. Insome embodiments, the biomass comprises, but is not limited tocultivated crops (e.g., grasses, including C4 grasses, such as switchgrass, cord grass, rye grass, miscanthus, reed canary grass, or anycombination thereof), sugar processing residues, for example, but notlimited to, bagasse (e.g., sugar cane bagasse, beet pulp [e.g., sugarbeet], or a combination thereof), agricultural residues (e.g., soybeanstover, corn stover, corn fiber, rice straw, sugar cane straw, rice,rice hulls, barley straw, corn cobs, wheat straw, canola straw, oatstraw, oat hulls, corn fiber, hemp, flax, sisal, cotton, or anycombination thereof), fruit pulp, vegetable pulp, distillers' grains,forestry biomass (e.g., wood, wood pulp, paper pulp, recycled wood pulpfiber, sawdust, hardwood, such as aspen wood, softwood, or a combinationthereof). Furthermore, in some embodiments, the biomass comprisescellulosic waste material and/or forestry waste materials, including butnot limited to, paper and pulp processing waste, municipal paper waste,newsprint, cardboard and the like. In some embodiments, biomasscomprises one species of fiber, while in some alternative embodiments,the biomass comprises a mixture of fibers that originate from differentbiomasses. In some embodiments, the biomass also comprises transgenicplants that express ligninase and/or cellulase enzymes (See e.g., US2008/0104724 A1).

As used herein, “lignocellulose” refers to a matrix of cellulose,hemicellulose and lignin. Economic production of biofuels fromlignocellulosic biomass typically involves conversion of the celluloseand hemicellulose components to fermentable sugars, typicallymonosaccharides such as glucose (from the cellulose) and xylose andarabinose (from the hemicelluloses). Nearly complete conversion can beachieved by a chemical pretreatment of the lignocellulose followed byenzymatic hydrolysis with cellulase enzymes. The chemical pretreatmentstep renders the cellulose more susceptible to enzymatic hydrolysis and,in some cases, also hydrolyzes the hemicellulose component. Numerouschemical pretreatment processes are known in the art, and include, butare not limited to, mild acid pretreatment at high temperatures anddilute acid, ammonium pretreatment or organic solvent extraction.

Lignin is a more complex and heterogeneous biopolymer than eithercellulose or hemicellulose and comprises a variety of phenolic subunits.Enzymatic lignin depolymerization can be accomplished by ligninperoxidases, manganese peroxidases, laccases and cellobiosedehydrogenases (CDH), often working in synergy. However, as the namesuggests, CDH enzymes also oxidize cellobiose to cellobionolactone.Several reports indicate that the oxidation of cellobiose by CDHenhances the rate of cellulose hydrolysis by cellulases by virtue ofreducing the concentrations of cellobiose, which is a potent inhibitorof some cellulase components (Mansfield et al., Appl. Environ.Microbiol., 63: 3804-3809 [1997]; and Igarishi et al., Eur. J. Biochem.,253: 101-106 [1998]). Recently, it has been reported that CDHs canenhance the activity of cellulolytic enhancing proteins from GlycosylHydrolase family 61 (See e.g., WO2010/080532A1).

As used herein, the term “lignocellulosic biomass” refers to any plantbiomass comprising cellulose and hemicellulose, bound to lignin

In some embodiments, the biomass is optionally pretreated to increasethe susceptibility of cellulose to hydrolysis by chemical, physical andbiological pretreatments (such as steam explosion, pulping, grinding,acid hydrolysis, solvent exposure, and the like, as well as combinationsthereof). Various lignocellulosic feedstocks find use, including thosethat comprise fresh lignocellulosic feedstock, partially driedlignocellulosic feedstock, fully dried lignocellulosic feedstock, and/orany combination thereof. In some embodiments, lignocellulosic feedstockscomprise cellulose in an amount greater than about 20%, more preferablygreater than about 30%, more preferably greater than about 40% (w/w).For example, in some embodiments, the lignocellulosic material comprisesfrom about 20% to about 90% (w/w) cellulose, or any amount therebetween,although in some embodiments, the lignocellulosic material comprisesless than about 19%, less than about 18%, less than about 17%, less thanabout 16%, less than about 15%, less than about 14%, less than about13%, less than about 12%, less than about 11%, less than about 10%, lessthan about 9%, less than about 8%,less than about 7%, less than about6%, or less than about 5% cellulose (w/w). Furthermore, in someembodiments, the lignocellulosic feedstock comprises lignin in an amountgreater than about 10%, more typically in an amount greater than about15% (w/w). In some embodiments, the lignocellulosic feedstock comprisessmall amounts of sucrose, fructose and/or starch. The lignocellulosicfeedstock is generally first subjected to size reduction by methodsincluding, but not limited to, milling, grinding, agitation, shredding,compression/expansion, or other types of mechanical action. Sizereduction by mechanical action can be performed by any type of equipmentadapted for the purpose, for example, but not limited to, hammer mills,tub-grinders, roll presses, refiners and hydrapulpers. In someembodiments, at least 90% by weight of the particles produced from thesize reduction have lengths less than between about 1/16 and about 4 in(the measurement may be a volume or a weight average length). In someembodiments, the equipment used to reduce the particle size is a hammermill or shredder. Subsequent to size reduction, the feedstock istypically slurried in water, as this facilitates pumping of thefeedstock. In some embodiments, lignocellulosic feedstocks of particlesize less than about 6 inches do not require size reduction.

As used herein, the term “lignocellulosic feedstock” refers to any typeof lignocellulosic biomass that is suitable for use as feedstock insaccharification reactions.

As used herein, the term “pretreated lignocellulosic feedstock,” refersto lignocellulosic feedstocks that have been subjected to physicaland/or chemical processes to make the fiber more accessible and/orreceptive to the actions of cellulolytic enzymes, as described above.

As used herein, the terms “lignocellulose-competent,”“lignocellulose-utilizing” and like terms refer to an organism thatsecretes enzymes that participate in lignin breakdown and hydrolysis.For example, in some embodiments, lignocellulose-competent fungal cellssecrete one or more lignin peroxidases, manganese peroxidases, laccasesand/or cellobiose dehydrogenases (CDH). These extracellular enzymes,essential for lignin degradation, are often referred to as“lignin-modifying enzymes” or “LMEs.”

A biomass substrate is said to be “pretreated” when it has beenprocessed by some physical and/or chemical means to facilitatesaccharification. As described further herein, in some embodiments, thebiomass substrate is “pretreated,” or treated using methods known in theart, such as chemical pretreatment (e g., ammonia pretreatment, diluteacid pretreatment, dilute alkali pretreatment, or solvent exposure),physical pretreatment (e.g., steam explosion or irradiation), mechanicalpretreatment (e.g., grinding or milling) and biological pretreatment(e.g., application of lignin-solubilizing microorganisms) andcombinations thereof, to increase the susceptibility of cellulose tohydrolysis.

In some embodiments, the substrate is slurried prior to pretreatment. Insome embodiments, the consistency of the slurry is between about 2% andabout 30% and more typically between about 4% and about 15%. In someembodiments, the slurry is subjected to a water and/or acid soakingoperation prior to pretreatment. In some embodiments, the slurry isdewatered using any suitable method to reduce steam and chemical usageprior to pretreatment. Examples of dewatering devices include, but arenot limited to pressurized screw presses (See e.g., WO 2010/022511,incorporated herein by reference) pressurized filters and extruders.

In some embodiments, the pretreatment is carried out to hydrolyzehemicellulose, and/or a portion thereof present in lignocellulose tomonomeric pentose and hexose sugars (e.g., xylose, arabinose, mannose,galactose, and/or any combination thereof). In some embodiments, thepretreatment is carried out so that nearly complete hydrolysis of thehemicellulose and a small amount of conversion of cellulose to glucoseoccurs. In some embodiments, an acid concentration in the aqueous slurryfrom about 0.02% (w/w) to about 2% (w/w), or any amount therebetween, istypically used for the treatment of the cellulosic substrate. Anysuitable acid finds use in these methods, including but not limited to,hydrochloric acid, nitric acid, and/or sulfuric acid. In someembodiments, the acid used during pretreatment is sulfuric acid. Steamexplosion is one method of performing acid pretreatment of biomasssubstrates (See e.g., U.S. Pat. No. 4,461,648). Another method ofpretreating the slurry involves continuous pretreatment (i.e., thecellulosic biomass is pumped though a reactor continuously). Thismethods are well-known to those skilled in the art (See e.g., U.S. Pat.No. 7,754,457).

In some embodiments, alkali is used in the pretreatment. In contrast toacid pretreatment, pretreatment with alkali may not hydrolyze thehemicellulose component of the biomass. Rather, the alkali reacts withacidic groups present on the hemicellulose to open up the surface of thesubstrate. In some embodiments, the addition of alkali alters thecrystal structure of the cellulose so that it is more amenable tohydrolysis. Examples of alkali that find use in the pretreatmentinclude, but are not limited to ammonia, ammonium hydroxide, potassiumhydroxide, and sodium hydroxide. One method of alkali pretreatment isAmmonia Freeze Explosion, Ammonia Fiber Explosion or Ammonia FiberExpansion (“AFEX” process; See e.g., U.S. Pat. Nos. 5,171,592;5,037,663; 4,600,590; 6,106,888; 4,356,196; 5,939,544; 6,176,176;5,037,663; and 5,171,592). During this process, the cellulosic substrateis contacted with ammonia or ammonium hydroxide in a pressure vessel fora sufficient time to enable the ammonia or ammonium hydroxide to alterthe crystal structure of the cellulose fibers. The pressure is thenrapidly reduced, which allows the ammonia to flash or boil and explodethe cellulose fiber structure. In some embodiments, the flashed ammoniais then recovered using methods known in the art. In some alternativemethods, dilute ammonia pretreatment is utilized. The dilute ammoniapretreatment method utilizes more dilute solutions of ammonia orammonium hydroxide than AFEX (See e.g., WO 2009/045651 and US2007/0031953). This pretreatment process may or may not produce anymonosaccharides.

An additional pretreatment process for use in the present inventionincludes chemical treatment of the cellulosic substrate with organicsolvents, in methods such as those utilizing organic liquids inpretreatment systems (See e.g., U.S. Pat. No. 4,556,430). These methodshave the advantage that the low boiling point liquids easily can berecovered and reused. Other pretreatments, such as the Organosolv™process, also use organic liquids (See e.g., U.S. Pat. No. 7,465,791).Subjecting the substrate to pressurized water may also be a suitablepretreatment method (See e.g., Weil et al., Appl. Biochem. Biotechnol.,68: 21-40 [1997]). In some embodiments, the pretreated cellulosicbiomass is processed after pretreatment by any of several steps, such asdilution with water, washing with water, buffering, filtration, orcentrifugation, or any combination of these processes, prior toenzymatic hydrolysis, as is familiar to those skilled in the art.

The pretreatment produces a pretreated feedstock composition (e.g., a“pretreated feedstock slurry”) that contains a soluble componentincluding the sugars resulting from hydrolysis of the hemicellulose,optionally acetic acid and other inhibitors, and solids includingunhydrolyzed feedstock and lignin. In some embodiments, the solublecomponents of the pretreated feedstock composition are separated fromthe solids to produce a soluble fraction. In some embodiments, thesoluble fraction, including the sugars released during pretreatment andother soluble components (e.g., inhibitors), is then sent tofermentation. However, in some embodiments in which the hemicellulose isnot effectively hydrolyzed during the pretreatment one or moreadditional steps are included (e.g., a further hydrolysis step(s) and/orenzymatic treatment step(s) and/or further alkali and/or acid treatment)to produce fermentable sugars. In some embodiments, the separation iscarried out by washing the pretreated feedstock composition with anaqueous solution to produce a wash stream and a solids stream comprisingthe unhydrolyzed, pretreated feedstock. Alternatively, the solublecomponent is separated from the solids by subjecting the pretreatedfeedstock composition to a solids-liquid separation, using any suitablemethod (e.g., centrifugation, microfiltration, plate and framefiltration, cross-flow filtration, pressure filtration, vacuumfiltration, etc.). Optionally, in some embodiments, a washing step isincorporated into the solids-liquids separation. In some embodiments,the separated solids containing cellulose, then undergo enzymatichydrolysis with cellulase enzymes in order to convert the cellulose toglucose. In some embodiments, the pretreated feedstock composition isfed into the fermentation process without separation of the solidscontained therein. In some embodiments, the unhydrolyzed solids aresubjected to enzymatic hydrolysis with cellulase enzymes to convert thecellulose to glucose after the fermentation process. In someembodiments, the pretreated cellulosic feedstock is subjected toenzymatic hydrolysis with cellulase enzymes.

As used herein, the term “chemical treatment” refers to any chemicalpretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin.

As used herein, the term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulosic material.

As used herein, the term “mechanical pretreatment” refers to anymechanical means for treating biomass, including but not limited tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling)

As used herein, the term “biological pretreatment” refers to anybiological pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin from cellulosic material.

As used herein, the term “recovered” refers to the harvesting,isolating, collecting, or recovering of protein from a cell and/orculture medium. In the context of saccharification, it is used inreference to the harvesting of fermentable sugars produced during thesaccharification reaction from the culture medium and/or cells. In thecontext of fermentation, it is used in reference to harvesting thefermentation product from the culture medium and/or cells. Thus, aprocess can be said to comprise “recovering” a product of a reaction(such as a soluble sugar recovered from saccharification) if the processincludes separating the product from other components of a reactionmixture subsequent to at least some of the product being generated inthe reaction.

As used herein, “increasing” the yield of a product (such as afermentable sugar) from a reaction occurs when a particular component ofinterest is present during the reaction (e.g., enzyme) causes moreproduct to be produced, compared with a reaction conducted under thesame conditions with the same substrate and other substituents, but inthe absence of the component of interest (e.g., without enzyme).

As used herein, a reaction is said to be “substantially free” of aparticular enzyme if the amount of that enzyme compared with otherenzymes that participate in catalyzing the reaction is less than about2%, about 1%, or about 0.1% (wt/wt).

As used herein, “fractionating” a liquid (e.g., a culture broth) meansapplying a separation process (e.g., salt precipitation, columnchromatography, size exclusion, and filtration) or a combination of suchprocesses to provide a solution in which a desired protein (e.g., acellulase enzyme, and/or a combination thereof) comprises a greaterpercentage of total protein in the solution than in the initial liquidproduct.

As used herein, the term “enzymatic hydrolysis,” refers to thehydrolysis of a substrate by an enzyme. In some embodiments, thehydrolysis comprises methods in which at least one enzyme is contactedwith at least one substrate to produce an end product. In someembodiments, the enzymatic hydrolysis methods comprise at least onecellulase and at least one glycosidase enzyme and/or a mixtureglycosidases that act on polysaccharides, (e.g., cellulose), to convertall or a portion thereof to fermentable sugars. “Hydrolyzing” and/or“hydrolysis” of cellulose or other polysaccharide occurs when at leastsome of the glycosidic bonds between two monosaccharides present in thesubstrate are hydrolyzed, thereby detaching from each other the twomonomers that were previously bonded.

It is intended that the enzymatic hydrolysis be carried out with anysuitable type of enzyme(s) capable of hydrolyzing at least one substrateto at least one end-product. In some embodiments, the substrate iscellulose, while in some other embodiments, it is lignocelluloses, andin still further embodiments, it is another composition (e.g., starch).In some embodiments, the end-product comprises at least one fermentablesugar. It is further intended that the enzymatic hydrolysis encompassprocesses carried out with any suitable type of cellulase enzymescapable of hydrolyzing the cellulose to glucose, regardless of theirsource. It is intended that any suitable source of enzyme will find usein the present invention, including but not limited to enzymes obtainedfrom fungi, such as Trichoderma spp., Aspergillus spp., Hypocrea spp.,Humicola spp., Neurospora spp., Orpinomyces spp., Gibberella spp.,Emericella spp., Chaetomium spp., Chrysosporium spp., Fusarium spp.,Penicillium spp., Magnaporthe spp., Phanerochaete spp., Trametes spp.,Lentinula edodes, Gleophyllum trabeiu, Ophiostoma piliferum, Corpinuscinereus, Geomyces pannorum, Cryptococcus laurentii, Aureobasidiumpullulans, Amorphotheca resinae, Leucosporidium scotti, Cunninghamellaelegans, Thermomyces lanuginosus, Myceliopthora thermophila, andSporotrichum thermophile, as well as those obtained from bacteria of thegenera Bacillus, Thermomyces, Clostridium, Streptomyces andThermobifida.

In some embodiments, the enzymatic hydrolysis is carried out at a pH andtemperature that is at or near the optimum for the cellulase enzymesbeing used. For example, in some embodiments, the enzymatic hydrolysisis carried out at about 30° C. to about 75° C., or any suitabletemperature therebetween, for example a temperature of about 30° C.,about 35° C., about 40° C., about 45° C., about 50° C., about 55° C.,about 60° C., about 65° C., about 70° C., about 75° C., or anytemperature therebetween, and a pH of about 3.5 to about 7.5, or any pHtherebetween (e.g., about 3.5, about 4.0, about 4.5, about 5.0, about5.5, about 6.0, about 6.5, about 7.0, about 7.5, or any suitable pHtherebetween). In some embodiments, the initial concentration ofcellulose, prior to the start of enzymatic hydrolysis, is preferablyabout 0.1% (w/w) to about 20% (w/w), or any suitable amount therebetween(e.g., about 0.1%, about 0.5%, about 1%, about 2%, about 4%, about 6%,about 8%, about 10%, about 12%, about 14%, about 15%, about 18%, about20%, or any suitable amount therebetween). In some embodiments, thecombined dosage of all cellulase enzymes is about 0.001 to about 100 mgprotein per gram cellulose, or any suitable amount therebetween (e.g.,about 0.001, about 0.01, about 0.1, about 1, about 5, about 10, about15, about 20, about 25, about 30, about 40, about 50, about 60, about70, about 80, about 90, about 100 mg protein per gram cellulose or anyamount therebetween). The enzymatic hydrolysis is carried out for anysuitable time period. In some embodiments, the enzymatic hydrolysis iscarried out for a time period of about 0.5 hours to about 200 hours, orany time therebetween (e.g., about 2 hours to about 100 hours, or anysuitable time therebetween). For example, in some embodiments, it iscarried out for about 0.5, about 1, about 2, about 5, about 7, about 10,about 12, about 14, about 15, about 20, about 25, about 30, about 35,about 40, about 45, about 50, about 55, about 60, about 65, about 70,about 75, about 80, about 85, about 90, about 95, about 100, about 120,about 140, about 160, about 180, about 200, or any suitable timetherebetween.

In some embodiments, the enzymatic hydrolysis is batch hydrolysis,continuous hydrolysis, and/or a combination thereof. In someembodiments, the hydrolysis is agitated, unmixed, or a combinationthereof. The enzymatic hydrolysis is typically carried out in ahydrolysis reactor. The cellulase enzyme composition is added to thepretreated lignocellulosic substrate prior to, during, or after theaddition of the substrate to the hydrolysis reactor. Indeed it is notintended that reaction conditions be limited to those provided herein,as modifications are well-within the knowledge of those skilled in theart. In some embodiments, following cellulose hydrolysis, any insolublesolids present in the resulting lignocellulosic hydrolysate, includingbut not limited to lignin, are removed using conventional solid-liquidseparation techniques prior to any further processing. In someembodiments, these solids are burned to provide energy for the entireprocess.

As used herein, the “total available cellulose” is the amount (wt %) ofcellulose that is accessible to enzymatic hydrolysis. Total availablecellulose is typically equal to, or very close to being equal to, theamount of initial cellulose present in a hydrolysis reaction.

As used herein, the “residual cellulose” is the portion (wt %) of thetotal available cellulose in the hydrolysis mixture that remainsunhydrolyzed. Residual cellulose can be measured using any suitablemethod known in the art. For example, it can be directly measured usingIR spectroscopy, or it can be measured by determining the amount ofglucose generated by concentrated acid hydrolysis of the residualsolids.

As used herein, the “total hydrolyzed cellulose” is the portion of thetotal available cellulose that is hydrolyzed in the hydrolysis mixture.For example, the total hydrolyzed cellulose can be calculated as thedifference between the “total available cellulose” and the “residualcellulose.”

As used herein, the “theoretical maximum glucose yield” is the maximumamount (wt %) of glucose that could be produced under given conditionsfrom the total available cellulose.

As used herein, “Gmax” refers to the maximum amount (wt %) of glucosethat could be produced from the total hydrolyzed cellulose. Gmax can becalculated, for example, by directly measuring the amount of residualcellulose remaining at the end of a reaction under a given reactionconditions, subtracting the amount of residual cellulose from the totalavailable cellulose to determine the total hydrolyzed cellulose, andthen calculating the amount of glucose that could be produced from thetotal hydrolyzed cellulose.

It will be appreciated by those skilled in the art that when calculatingtheoretical values such as Gmax and theoretical maximum glucose yield,the mass of two hydrogen atoms and one oxygen atom that are added to theglucose molecule in the course of the hydrolysis reaction are taken intoaccount. For example, when a polymer of “n” glucose units is hydrolyzed,(n−1) units of water are added to the glucose molecules formed in thehydrolysis, so the weight of the glucose produced is about 10% greaterthan the weight of cellulose consumed in the hydrolysis (e.g.,hydrolysis of 1 g cellulose would produce about 1.1 g glucose).

Thus, as an example, where 5 g of total available cellulose is presentat the beginning of a hydrolysis reaction, and 2 g of residual celluloseremains after the reaction, the total hydrolyzed cellulose is 3 gcellulose. A theoretical maximum glucose yield of 100% (w/w) under thereaction conditions is about 5.5 g of glucose. Gmax is calculated basedon the 3 g of cellulose that was released or converted in the reactionby hydrolysis. Thus, in this example, a Gmax of 100% (w/w) is about 3.3g of glucose. Cellulose levels, either the total available amountpresent in the substrate or the amount of unhydrolyzed or residualcellulose, can be quantified by any of a variety of methods known in theart, such as by IR spectroscopy or by measuring the amount of glucosegenerated by concentrated acid hydrolysis of the cellulose (See e.g.,U.S. Pat. Nos. 6,090,595 and 7,419,809).

As used herein, the term “undissolved solids” refers to solid materialwhich is suspended, but not dissolved, in a liquid. As is well known inthe art, the concentration of suspended or undissolved solids can bedetermined by any suitable method (e.g., by filtering a sample of theslurry using glass microfiber filter paper, washing the filter cake withwater, and drying the cake overnight at about 105° C.).

As used herein, the terms “unhydrolyzed solids,” “unconverted solids,”and the like refer to cellulose that is not digested by the cellulaseenzyme(s), as well as non-cellulosic, or other, materials that are inertto the cellulase enzyme(s), present in the feedstock.

As used herein, the term “by-product” refers to an organic molecule thatis an undesired product of a particular process (e.g.,saccharification).

As used herein, the term “antibodies” refers to immunoglobulins.Antibodies include but are not limited to immunoglobulins obtaineddirectly from any species from which it is desirable to obtainantibodies. In addition, the present invention encompasses modifiedantibodies. The term also refers to antibody fragments that retain theability to bind to the epitope that the intact antibody binds andincludes polyclonal antibodies, monoclonal antibodies, chimericantibodies, anti-idiotype (anti-ID) antibodies. Antibody fragmentsinclude, but are not limited to the complementarity-determining regions(CDRs), single-chain fragment variable regions (scFv), heavy chainvariable region (VH), and light chain variable region (VL) fragments.

As used herein, the terms “thermally stable” and “thermostable” refer toenzymes of the present invention that retain a specified amount ofenzymatic activity after exposure to identified temperatures over agiven period of time under conditions prevailing during the use of theenzyme, for example, when exposed to altered temperatures. “Alteredtemperatures” include increased or decreased temperatures. In someembodiments, the enzymes retain at least about 50%, about 60%, about70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%,about 96%, about 97%, about 98%, or about 99% enzymatic activity afterexposure to altered temperatures over a given time period, for example,at least about 60 minutes, about 120 minutes, about 180 minutes, about240 minutes, about 300 minutes, etc.

As used herein, the term “thermophilic fungus” refers to any funguswhich exhibits optimum growth at a temperature of at least about 37° C.,and generally below about 100° C., such as for example between about 37°C. to about 80° C., between about 37° C. to about 75° C., between about40° C. to about 65° C., or between about 40° C. to about 60° C.Typically, the optimum growth is exhibited at a temperature of at leastabout 40° to about 60° C.

As used herein, “solvent stable” refers to a polypeptide that maintainssimilar activity (more than for example, about 60% to about 80%) afterexposure to varying concentrations (e.g., about 5 to about 99%) of anon-aqueous solvent (e.g., isopropyl alcohol, tetrahydrofuran,2-methyltetrahydrofuran, acetone, toluene, butylacetate, methyltert-butylether, etc.) for a period of time (e.g., about 0.5 to about 24hrs) compared to a reference polypeptide.

As used herein, the term “oxidation stable” refers to enzymes of thepresent invention that retain a specified amount of enzymatic activityover a given period of time under conditions prevailing during the useof the invention, for example while exposed to or contacted withoxidizing agents. In some embodiments, the enzymes retain at least about50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%,about 92%, about 95%, about 96%, about 97%, about 98%, or about 99%enzymatic activity after contact with an oxidizing agent over a giventime period, for example, at least about 1 minute, about 3 minutes,about 5 minutes, about 8 minutes, about 12 minutes, about 16 minutes,about 20 minutes, etc.

As used herein, “pH stable” refers to a polypeptide that maintainssimilar activity (more than for example, about 60% to about 80%) afterexposure to low or high pH (e.g., about 4.5 to about 6 or about 8 toabout 12) for a period of time (e.g., 0.5-24 hrs) compared to areference polypeptide.

As used herein, the term “enhanced stability” in the context of anoxidation, chelator, thermal and/or pH stable enzyme refers to a higherretained enzymatic activity over time as compared to other enzymesand/or wild-type enzymes.

As used herein, the term “diminished stability” in the context of anoxidation, chelator, thermal and/or pH stable enzyme refers to a lowerretained enzymatic activity over time as compared to other enzymesand/or wild-type enzymes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved fungal strains. In someembodiments, the improved fungal strain finds use in hydrolyzingcellulosic material to glucose. As indicated herein, the presentinvention provides fungal strains that have reduced secreted activity ofan endogenous cellobiose dehydrogenase. In some embodiments, the fungalstrains secrete enzyme mixtures that improve the yield of fermentablesugars from cellulose. Previous reports have indicated that theoxidation of cellobiose by cellobiose dehydrogenase enhances the rate ofcellulose hydrolysis by cellulases. In contrast to the traditionalthinking in the art, the present invention surprisingly provides genomicmodifications that reduce cellobiose dehydrogenase activity and resultin improvement in the yield of fermentable sugars from cellulose.Advantageously, the genetically modified cellulase-producing fungalcells provided herein secrete enzyme mixtures that result in improvedyields of fermentable sugars such as glucose from cellulose.

Lignocellulose (also “lignocellulosic biomass”) comprises a matrix ofcellulose, hemicellulose and lignin. Economic production of biofuelsfrom lignocellulosic biomass typically involves conversion of thecellulose and hemicellulose components to fermentable sugars, typicallymonosaccharides such as glucose (from the cellulose) and xylose andarabinose (from the hemicelluloses). Nearly complete conversion can beachieved by a chemical pretreatment of the lignocellulose followed byenzymatic hydrolysis with cellulase enzymes. The chemical pretreatmentstep renders the cellulose more susceptible to enzymatic hydrolysis andin some cases, also hydrolyzes the hemicellulose component. Numerouschemical pretreatment processes known in the art find use in the presentinvention, and include, but are not limited to, mild acid pretreatmentat high temperatures and dilute acid, ammonium pretreatment and/ororganic solvent extraction.

Cellulase is typically a mixture of different types of cellulolyticenzymes (e.g., endoglucanases and cellobiohydrolases, the latter arealso referred to as “exoglucanases”) that act synergistically to breakdown the cellulose to soluble di- or oligosaccharides such ascellobiose, which are then further hydrolyzed to glucose bybeta-glucosidase. Cellulase enzymes are produced by a wide variety ofmicroorganisms. Cellulases, as well as hemicellulases from filamentousfungi and some bacteria are widely exploited for many industrialapplications that involve processing of natural fibers to sugars.

Lignin is a more complex and heterogeneous biopolymer than eithercellulose or hemicellulose and comprises a variety of phenolic subunits.Enzymatic lignin depolymerization can be accomplished by ligninperoxidases, manganese peroxidases, laccases, and/or cellobiosedehydrogenases (CDH), often working in synergy. However, as the namesuggests, CDH enzymes also oxidize cellobiose to cellobionolactone.Several reports indicate that the oxidation of cellobiose by CDHenhances the rate of cellulose hydrolysis by cellulases by virtue ofreducing the concentrations of cellobiose, which is a potent inhibitorof some cellulase components (See e.g., Mansfield et al., Appl. Environ.Microbiol., 63: 3804-3809 [1997]; and Igarishi et al., Eur. J. Biochem.,253:101-106 [1998]). Recently, it has been reported that CDHs canenhance the activity of cellulolytic enhancing proteins from GlycosylHydrolase family 61 (See e.g., WO2010/080532A1).

Among the cellulase-producing filamentous fungi, there are those thatalso produce a variety of enzymes involved in lignin degradation. Forexample, organisms of such genera as Myceliophthora, Chrysosporium,Sporotrichum, Thielavia, Phanerochaete and Trametes produce and secretea mixture of cellulases, hemicellulases and lignin degrading enzymes.These types of organisms are commonly called “white rot fungi” by virtueof their ability to digest lignin and to distinguish them from the“brown rot” fungi (such as Trichoderma) which typically cannot digestlignin.

Genetically Modified Fungal Cells

The genetically modified fungal cells provided herein permit a reductionin the amount of endogenous cellobiose dehydrogenase activity that issecreted by the cell. In some genetically modified fungal cells providedherein, the cellobiose dehydrogenase activity secreted by the cell isreduced by at least about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more, relative to the level ofcellobiose dehydrogenase activity secreted by the unmodified parentalfungal cell grown or cultured under essentially the same cultureconditions. In some embodiments, a genetically modified fungal cellprovides at least about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99% or more, relative to the level of cellobiosedehydrogenase activity secreted by the unmodified parental fungal cellgrown or cultured under essentially the same conditions.

It will be readily appreciated that any suitable genetic modificationknown in the art can be employed to reduce the secreted activity of theendogenous cellobiose dehydrogenase. For example, as described below,modifications contemplated herein include modifications that reduce theamount of cellobiose dehydrogenase secreted by the cell. Modificationsthat reduce the amount of cellobiose dehydrogenase expressed by the cellare also contemplated. Additional embodiments include modifications thatreduce the transcription level of cellobiose dehydrogenase. Stillfurther embodiments include the complete or partial deletion of a geneencoding cellobiose dehydrogenase. Other embodiments includemodifications that reduce the catalytic efficiency of cellobiosedehydrogenase.

Secreted Enzymes

In some embodiments, the fungal cells of the present invention have beengenetically modified to reduce the amount of the endogenous cellobiosedehydrogenase secreted by the cell. A reduction in the amount ofsecreted cellobiose dehydrogenase can be a complete or partial reductionof the cellobiose dehydrogenase secreted to the extracellular milieuReduction in the amount of secreted cellobiose dehydrogenase can beaccomplished by reducing the amount of cellobiose dehydrogenase producedby the cell and/or by reducing the ability of the cell to secrete thecellobiose dehydrogenase produced by the cell. Methods for reducing theability of the cell to secrete a polypeptide can be performed accordingto any of a variety of suitable methods known in the art (See e.g., Fassand Engels J. Biol. Chem., 271:15244-15252 [1996], which is incorporatedby reference herein in its entirety). For example, the gene encoding asecreted polypeptide can be modified to delete or inactivate a secretionsignal peptide. In some embodiments, the fungal cells have beengenetically modified to disrupt the N-terminal secretion signal peptideof the cellobiose dehydrogenase. In some embodiments, the amount ofcellobiose dehydrogenase secreted by the cell is reduced by at leastabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or more, relative to the secretion of cellobiosedehydrogenase in an unmodified organism grown or cultured underessentially the same culture conditions.

Furthermore, in some embodiments, the total amount of cellobiosedehydrogenase activity is reduced by at least about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more, relativeto the total amount of cellobiose dehydrogenase secreted in anunmodified organism grown or cultured under essentially the same cultureconditions.

Decreased secretion of a cellobiose dehydrogenase can be determined byany of a variety of suitable methods known in the art for detection ofprotein or enzyme levels. For example, the levels of cellobiosedehydrogenase in the supernatant of a fungal culture can be detectedusing Western blotting techniques or any other suitable proteindetection techniques that use an antibody specific to cellobiosedehydrogenase. Similarly, secreted cellobiose dehydrogenase activity inthe supernatant of a fungal culture can be measured using assays forcellobiose dehydrogenase activity as described in greater detail herein.

Expression Level

In some embodiments, the fungal cells have been genetically modified toreduce the amount of the endogenous cellobiose dehydrogenase expressedby the cell. As used herein, expression refers to conversion of theinformation encoded in a gene to the protein encoded by that gene. Thus,a reduction of the amount of an expressed cellobiose dehydrogenaserepresents a reduction in the amount of the cellobiose dehydrogenasethat is eventually translated by the cell. In some such embodiments, thereduction in the expression is accomplished by reducing the amount ofmRNA that is transcribed from a gene encoding cellobiose dehydrogenase.In some other embodiments, the reduction in the expression isaccomplished by reducing the amount of protein that is translated from amRNA encoding cellobiose dehydrogenase.

The amount of cellobiose dehydrogenase expressed by the cell can bereduced by at least about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more, relative to the expression ofcellobiose dehydrogenase in an unmodified fungal cell. In some suchembodiments, the reduction in the expression is accomplished by reducingthe amount of mRNA that is transcribed from a gene encoding cellobiosedehydrogenase in an unmodified organism grown or cultured underessentially the same culture conditions.

Furthermore, in some embodiments, a reduction in the expression level ofa cellobiose dehydrogenase results in at least about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, 85% about, 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about a 99% reduction inthe total expression level of cellobiose dehydrogenase activity by thefungal cell relative to an unmodified fungal cell grown or culturedunder essentially the same culture conditions.

Decreased expression of a cellobiose dehydrogenase can be determined byany of a variety of methods known in the art for detection of protein orenzyme levels. For example, the levels of cellobiose dehydrogenase inthe supernatant of a fungal culture can be detected using Westernblotting techniques or any other suitable protein detection techniquesthat use an antibody specific to cellobiose dehydrogenase.

Methods for reducing expression of a polypeptide are well known and canbe performed using any of a variety of suitable methods known in theart. For example, the gene encoding a secreted polypeptide can bemodified to disrupt a translation initiation sequence such as aShine-Delgarno sequence or a Kozak consensus sequence. Furthermore, thegene encoding a secreted polypeptide can be modified to introduce aframeshift mutation in the transcript encoding the endogenous cellobiosedehydrogenase. It will also be recognized that usage of uncommon codonscan result in reduced expression of a polypeptide. It will beappreciated that in some embodiments, the gene encoding the cellobiosedehydrogenase has at least one nonsense mutation that results in thetranslation of a truncated protein.

Other methods of reducing the amount of expressed polypeptide includepost-transcriptional RNA silencing methodologies such as antisense RNAand RNA interference. Antisense techniques are well-established, andinclude using a nucleotide sequence complementary to the nucleic acidsequence of the gene. More specifically, expression of the gene by afungal cell may be reduced or eliminated by introducing a nucleotidesequence complementary to the nucleic acid sequence, which may betranscribed in the cell and is capable of hybridizing to the mRNAproduced in the cell. Under conditions allowing the complementaryanti-sense nucleotide sequence to hybridize to the mRNA, the amount ofprotein translated is thus reduced or eliminated. Methods for expressingantisense RNA are known in the art (See e.g., Ngiam et al., Appl EnvironMicrobiol., 66(2):775-82 [2000]; and Zrenner et al., Planta.,190(2):247-52 [1993]), both of which are hereby incorporated byreference herein in their entirety).

Furthermore, modification, downregulation or inactivation of the genemay be obtained via RNA interference (RNAi) techniques (See e.g.,Kadotani et al. Mol. Plant Microbe Interact., 16:769-76 [2003], which isincorporated by reference herein in its entirety). RNA interferencemethodologies include double stranded RNA (dsRNA), short hairpin RNAs(shRNAs) and small interfering RNAs (siRNAs). Potent silencing usingdsRNA may be obtained using any suitable technique (See e.g., Fire etal., Nature 391:806-11 [1998]). Silencing using shRNAs is alsowell-established (See e.g., Paddison et al., Genes Dev., 16:948-958[2002]). Silencing using siRNA techniques are also known (See e.g.,Miyagishi et al., Nat. Biotechnol., 20:497-500 [2002]). The content ofeach of the above-cited references is incorporated by reference hereinin its entirety.

Transcription Level

In some embodiments, the fungal cells of the present invention have beengenetically modified to reduce the transcription level of a geneencoding the endogenous cellobiose dehydrogenase. As used herein,transcription and similar terms refer to the conversion of theinformation encoded in a gene to an RNA transcript. Accordingly, areduction of the transcription level of a cellobiose dehydrogenase is areduction in the amount of RNA transcript of an RNA coding for acellobiose dehydrogenase. In some embodiments, the transcription levelis reduced by at least about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more, relative to the transcriptionlevel of a cellobiose dehydrogenase in an unmodified organism grown orcultured under essentially the same culture conditions.

Furthermore, in some embodiments, a reduction in the transcription levelof a cellobiose dehydrogenase results in at least about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about a 99%reduction in the total cellobiose dehydrogenase secreted by the fungalcell relative to an unmodified organism grown or cultured underessentially the same culture conditions. Decreased transcription can bedetermined by any of a variety of methods known in the art for detectionof transcription levels. For example, the levels of transcription of aparticular mRNA in a fungal cell can be detected using quantitativeRT-PCR techniques or other RNA detection techniques that specificallydetect a particular mRNA. Methods for reducing transcription level of agene can be performed according to any suitable method known in the art,and include partial or complete deletion of the gene, and disruption orreplacement of the promoter of the gene such that transcription of thegene is greatly reduced or even inhibited. For example, the promoter ofthe gene can be replaced with a weak promoter (See e.g., U.S. Pat. No.6,933,133, which is incorporated by reference herein in its entirety).Thus, where the weak promoter is operably linked with the codingsequence of an endogenous polypeptide, transcription of that gene isgreatly reduced or inhibited.

Gene Deletion

In some embodiments, the fungal cells of the present invention have beengenetically modified to at least partially delete a gene encoding theendogenous cellobiose dehydrogenase. Typically, this deletion reduces oreliminates the total amount of endogenous cellobiose dehydrogenasesecreted by the fungal cell. In some embodiments, complete ornear-complete deletion of the gene sequence is contemplated. However, adeletion mutation need not completely remove the entire gene sequenceencoding cellobiose dehydrogenase, in order to reduce the amount ofendogenous cellobiose dehydrogenase secreted by the fungal cell. Forexample, in some embodiments, there is a partial deletion that removesone or more nucleotides encoding an amino acid in a cellobiosedehydrogenase active site, encoding a secretion signal, or encodinganother portion of the cellobiose dehydrogenase that plays a role inendogenous cellobiose dehydrogenase activity being secreted by thefungal cell.

A deletion in a gene encoding cellobiose dehydrogenase in accordancewith the embodiments provided herein include a deletion of one or morenucleotides in the gene encoding the cellobiose dehydrogenase. In someembodiments, there is a deletion of at least about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, ofthe gene encoding the cellobiose dehydrogenase, wherein the amount ofcellobiose dehydrogenase secreted by the cell is reduced.

Thus, in some embodiments, the deletion results in at least about 5%,about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about a99% reduction in the activity of the cellobiose dehydrogenase secretedby the fungal cell, relative to the activity of cellobiose dehydrogenasesecreted by an unmodified organism grown or cultured under essentiallythe same culture conditions.

Furthermore, in some embodiments, the deletion results in at least about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout a 99% reduction in the total cellobiose dehydrogenase secreted bythe fungal cell relative to an unmodified fungal cell grown or culturedunder essentially the same culture conditions.

Deletion of a cellobiose dehydrogenase gene can be detected andconfirmed by any of a variety of methods known in the art for detectionof gene deletions, including the methods provide herein and in theExamples. For example, as exemplified herein, gene deletion can beconfirmed using PCR amplification of the modified genomic region. Itwill be appreciated that additional suitable techniques for confirmingdeletion can be used and are well known, including Southern blottechniques, DNA sequencing of the modified genomic region, and screeningfor positive or negative markers incorporated during recombinationevents.

Methods for complete and/or partial deletion of a gene are well-knownand the genetically modified fungal cells described herein can begenerated using any of a variety of deletion methods known in the artthat result in a reduction in the amount of endogenous cellobiosedehydrogenase secreted by the cells. Such methods may advantageouslyinclude standard gene disruption using homologous flanking markers (Seee.g., Rothstein, Meth. Enzymol., 101:202-211 [1983], incorporated hereinby reference in its entirety). Additional techniques for gene deletioninclude PCR-based methods for standard deletion (See e.g., Davidson etal., Microbiol., 148:2607-2615 [2002], incorporated herein by referencein its entirety).

Additional gene deletion techniques include “positive-negative”cassettes; cre/lox based deletion, biolistic transformation to increasehomologous recombination, and Agrobacterium-mediated gene disruption.The “positive-negative” method employs cassettes which consist of onemarker gene for positive screening and another marker gene for negativescreening (See e.g., Chang et al., Proc. Natl. Acad. Sci. USA84:4959-4963 [1987]). Cre/lox based methodologies employ elimination ofmarker genes using expression of Cre recombinase (See e.g., Florea etal., Fung. Genet. Biol., 46:721-730 [2009]).

Methods to introduce DNA or RNA into fungal cells are known to those ofskill in the art and include, but are not limited to PEG-mediatedtransformation of protoplasts, electroporation, biolistictransformation, and Agrobacterium-mediated transformation. Biolistictransformation employs a process in which DNA or RNA is introduced intocells on micron-sized particles, thus increasing delivery of a deletionconstruct to the fungal cell (See e.g., Davidson et al., Fung. Genet.Biol., 29:38-48 [2000]). Similarly, Agrobacterium-mediatedtransformation in conjunction with linear or split-marker deletioncassettes can facilitate delivery of deletion constructs to the targetcell (See e.g., Wang et al., Curr. Genet., 56:297-307 [2010]).

Further methods for complete or partial gene deletion include disruptionof the gene. Such gene disruption techniques are known to those of skillin the art, including, but not limited to insertional mutagenesis, theuse of transposons, and marked integration. However, it will beappreciated that any suitable technique that provides for disruption ofthe coding sequence or any other functional aspect of a gene finds usein generating the genetically modified fungal cells provided herein.Methods of insertional mutagenesis can be performed according to anysuitable method known in the art (See e.g., Combier et al., FEMSMicrobiol Lett., 220:141-8 [2003], which is incorporated by referenceherein in its entirety). In addition, Agrobacterium-mediated insertionalmutagenesis can be used to insert a sequence that disrupts the functionof the encoded gene, such as disruption of the coding sequence or anyother functional aspect of the gene.

Transposon mutagenesis methodologies provide another means for genedisruption. Transposon mutagenesis is well known in the art, and can beperformed using in vivo techniques (See e.g., Firon et al., Eukaryot.Cell 2:247-55 [2003]); or by the use of in vitro techniques (See e.g.,Adachi et al., Curr. Genet., 42:123-7 [2002]) ; both of these referencesare incorporated by reference in their entireties. Thus, targeted genedisruption using transposon mutagenesis can be used to insert a sequencethat disrupts the function of the encoded gene, such as disruption ofthe coding sequence or any other functional aspect of the gene.

Restriction enzyme-mediated integration (REMI) is another methodologyfor gene disruption, and is well known in the art (See e.g., Thon etal., Mol. Plant Microbe Interact., 13:1356-65 [2000], which isincorporated by reference herein in its entirety). REMI generatesinsertions into genomic restriction sites in an apparently randommanner, some of which cause mutations. Thus, insertional mutants thatdemonstrate a disruption in the gene encoding the endogenous cellobiosedehydrogenase can be selected and utilized as provided herein.

Catalytic Disruption

In some other embodiments, the fungal cell has been genetically modifiedto reduce the catalytic efficiency of the cellobiose dehydrogenase. Areduction in catalytic efficiency refers to a reduction in the activityof cellobiose dehydrogenase, relative to unmodified cellobiosedehydrogenase, as measured using standard techniques, as provided hereinor otherwise known in the art. Thus, a genetic modification that reducescatalytic efficiency can result in, for example, a translated proteinproduct that has a reduction in enzymatic activity.

A reduction in catalytic efficiency is a reduction of cellobiosedehydrogenase activity of about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or more, relative to unmodifiedcellobiose dehydrogenase, as measured using standard techniques. In somefurther embodiments, the genetic modification results in a reduction ofcellobiose dehydrogenase activity of at least about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% in the totalcellobiose dehydrogenase activity secreted by the fungal cell, ascompared to unmodified cellobiose dehydrogenase, as measured usingstandard techniques.

Methods for reducing catalytic efficiency of dehydrogenases are wellknown, and as such, any of a variety of suitable methods known in theart for reducing catalytic efficiency find use in genetically modifyingthe fungal cells provided herein. Thus, for example, the fungal cell canbe genetically modified to inactivate one or more residues in an activesite of the cellobiose dehydrogenase (See e.g., Frederik et at.,Biochem., 42:4049-4056 [2003], incorporated by reference herein in itsentirety). For example, one or more residues can be modified to decreasesubstrate binding, and/or one or more residues can be modified todecrease the catalytic activity of the cellobiose dehydrogenase.Accordingly, one or more residues in the electron acceptor (e.g.,flavin) binding domain, or any substrate binding domain of cellobiosedehydrogenase can be performed to reduce or inactivate the catalyticefficiency of the cellobiose dehydrogenase. Similarly, it will beapparent that mutation of residues outside an active site can result inallosteric change in the shape or activity of the cellobiosedehydrogenase, such that the catalytic efficient of the enzyme isreduced.

In some embodiments, other domains are targeted for at least onemutation which results in reducing catalytic efficiency of theendogenous cellobiose dehydrogenase. For example, in some embodiments, amutation to one or more residues in a heme-binding domain of cellobiosedehydrogenase can result in reduced catalytic efficiency (See e.g.,Rotsaert et al., Arch. Biochem. Biophys., 390:206-14 [2001], which isincorporated by reference herein in its entirety).

Fungal Cells

As indicated herein, the present invention provides fungal cells fromthe family Chaetomiaceae that have been genetically modified to reducethe amount of endogenous cellobiose dehydrogenase activity that issecreted by the cell, where the fungal cell is capable of secreting acellulase-containing enzyme mixture. The Chaetomiaceae are a family offungi in the Ascomycota, class Sordariomycetes. The family Chaetomiaceaeincludes the genera Achaetomium, Aporothielavia, Chaetomidium,Chaetomium, Corylomyces, Corynascus, Farrowia, Thielavia, Zopfiella, andMyceliophthora. In some embodiments, the genetically modified fungalcell provided herein is a Chaetomiaceae family member selected fromMyceliophthora, Thielavia, Corynascus, and Chaetomium.

In some embodiments, the genetically modified fungal cell is an anamorphor teleomorph of a Chaetomiaceae family member selected fromMyceliophthora, Thielavia, Corynascus, and Chaetomium. In someembodiments, the genetically modified fungal cell is selected fromSporotrichum, Chrysosporium, Paecilomyces, Talaromyces and Acremonium.It is also contemplated that the genetically modified fungal cell canalso be selected from the genera Ctenomyces, Thermoascus, andScytalidium, including anamorphs and teleomorphs of fungal cells ofthese genera. In some embodiments, the genetically modified fungal cellis selected from the strains of Sporotrichum cellulophilum, Thielaviaheterothallica, Corynascus heterothallicus, Thielavia terrestris, andMyceliophthora thermophila, including anamorphs and teleomorphs thereof.It is not intended that the present invention be limited to anyparticular genus within the Chaetomiaceae family In some furtherembodiments, the genetically modified fungal cell is a thermophilicspecies of Acremonium, Arthroderma, Corynascus, Thielavia,Myceliophthora, Thermoascus, Chromocleista, Byssochlamys, Sporotrichum,Chaetomium, Chrysosporium, Scytalidium, Ctenomyces, Paecilomyces, orTalaromyces. It will be understood that for all of the aforementionedspecies, the genetically modified fungal cell presented hereinencompasses both the perfect and imperfect states, and other taxonomicequivalents (e.g., anamorphs), regardless of the species name by whichthey are known (See e.g., Cannon, Mycopathol., 111:75-83 [1990];Moustafa et al., Persoonia 14:173-175 [1990]; Upadhyay et al.,Mycopathol., 87:71-80 [1984]; Guarro et al., Mycotaxon 23: 419-427[1985]; Awao et al., Mycotaxon 16:436-440 [1983]; and von Klopotek,Arch. Microbiol., 98:365-369 [1974]). Those skilled in the art willreadily recognize the identity of appropriate equivalents. Accordingly,it will be understood that, unless otherwise stated, the use of aparticular species designation in the present disclosure also refers tospecies that are related by anamorphic or teleomorphic relationship. Forexample, the following species are anamorphs or teleomorphs and maytherefore be considered as synonymous: Myceliophthora thermophila,Sporotrichum thermophile, Sporotrichum thermophilum, Sporotrichumcellulophilum, Chrysosporium thermophile, Corynascus heterothallicus,and Thielavia heterothallica.

In some embodiments, the genetically modified fungal cells providedherein are cellulase-producing fungal cells. In some embodiments, thecellulase-producing fungal cells express and secrete a mixture ofcellulose hydrolyzing enzymes. In some embodiments, the geneticallymodified fungal cells provided herein are fungal cells from the familyChaetomiaceae that secrete two or more cellulose hydrolyzing enzymes(e.g., endoglucanase, cellobiohydrolase, and/or beta-glucosidase). Insome additional embodiments, the cellulase-producing fungal cellsproduce two or more of these enzymes, in any combination.

Additionally, in some embodiments, the genetically modified fungal cellis derived from a lignocellulose-competent parental fungal cell. In someembodiments, lignocellulose-competent fungal cells secrete one or morelignin peroxidases, manganese peroxidases, laccases and/or cellobiosedehydrogenases (CDH).

The present invention also provides a fungal culture in a vesselcomprising a genetically modified fungal cell as described hereinabove.In some embodiments, the vessel comprises a liquid medium, such asfermentation medium. For example, the vessel can be a flask, bioprocessreactor, or any suitable container. In some embodiments, the vesselcomprises a solid growth medium. For example, the solid medium can be anagar medium such as potato dextrose agar, carboxymethylcellulose,cornmeal agar, and any other suitable medium. In some embodiments, thefungal cell described hereinabove is an isolated fungal cell.

Cellobiose Dehydrogenase

As indicated herein, the terms “cellobiose dehydrogenase” and “CDH”refer to a cellobiose: acceptor 1-oxidoreductase that catalyzes theconversion of cellobiose in the presence of an acceptor tocellobiono-1,5-lactone and a reduced acceptor. Examples of cellobiosedehydrogenases fall into the enzyme classification (E.C. 1.1.99.18).Typically 2,6-dichloroindophenol can act as acceptor, as can iron,especially Fe(SCN)₃, molecular oxygen, ubiquinone, or cytochrome C, andother polyphenolics, such as lignin. Substrates of the enzyme includecellobiose, cello-oligosaccharides, lactose, andD-glucosyl-1,4-β-D-mannose, glucose, maltose, mannobiose,thiocellobiose, galactosyl-mannose, xylobiose, xylose. Electron donorsinclude beta-1-4 dihexoses with glucose or mannose at the reducing end,though alpha-1-4 hexosides, hexoses, pentoses, and beta-1-4 pentomerscan act as substrates for at least some of these enzymes (See e..g,Henriksson et al., Biochim. Biophys. Acta Prot. Struct. Mol. Enzymol.,1383: 48-54 [1998]; and Schou et al., Biochem. J., 330: 565-571 [1998]).

In some embodiments, a CDH enzyme contains both the conservedglucose-methanol-choline (GMC) oxido-reductase N and the GMCoxido-reductase C domains. In some other embodiments, a CDH contains theGMC oxido-reductase N domain alone. The GMC oxidoreductases are FADflavoprotein oxidoreductases (See e.g., Cavener, J. Mol. Biol.,223:811-814 [1992]; and Vrielink and Blow, Biochem., 32:11507-15[1993]). The GMC oxidoreductases include a variety of proteins, such ascholine dehydrogenase, methanol oxidase, and cellobiose dehydrogenase(CDH), which share a number of regions with sequence similarities. Oneof these regions, located in the N-terminal section, corresponds to theFAD ADP-binding domain, as further defined by the Pfam database underthe entry GMC_oxred_N (PF00732). Similarly, the C-terminal conserveddomain (GMC oxido-reductase C domain) is defined as set forth in thePfam database under the entry GMC_oxred_C (PF05199).

Cellobiose dehydrogenases can be categorized into two families The firstfamily contains a catalytic portion and the second family contains acatalytic portion and a cellulose binding motif (CBM). The 3-dimensionalstructure of an exemplary cellobiose dehydrogenase features two globulardomains, each containing one of two cofactors, namely a heme or aflavin. The active site lies at a cleft between the two domains.Oxidation of cellobiose typically occurs via 2-electron transfer fromcellobiose to the flavin, generating cellobiono-1,5-lactone and reducedflavin. The active FAD is regenerated by electron transfer to the hemegroup, leaving a reduced heme. The native state heme is regenerated byreaction with the oxidizing substrate at the second active site. In someembodiments, the acceptor is preferentially iron ferricyanide,cytochrome C, or an oxidized phenolic compound such asdichloroindophenol (DCIP), an acceptor commonly used for colorimetricassays. Metal ions and 0₂ are also acceptors, but for most cellobiosedehydrogenases the reaction rate of cellobiose oxidase for theseacceptors is several orders of magnitude lower than that observed foriron or organic oxidants. Following cellobionolactone release, theproduct may undergo spontaneous ring-opening to generate cellobionicacid (See e.g., Hallberg et al., J. Biol. Chem., 278:7160-7166 [2003]).Those of skill in the art will appreciate that cellobiose dehydrogenaseenzyme activity typically employs the presence of oxygen or anequivalent redox acceptor, which may be, for example, lignin, molecularoxygen, cytochrome c, redox dyes, benzoquinones and/or Fe²⁺ complexes.

Cellobiose dehydrogenase activity can be measured using any of a varietyof suitable methods known in the art (See e.g., Schou et al., BiochemJ., 220:565-71 [1998], which is incorporated by reference in itsentirety). For example, DCPIP (2,6-dichlorophenolindophenol) reductionby CDH activity in the presence of cellobiose can be monitored byabsorbance at 530 nm

As provided herein, a fungal cell that has been genetically modified toreduce the secreted activity of a cellobiose dehydrogenase typically hasreduced secreted activity of an endogenous cellobiose dehydrogenase.Accordingly, one or more cellobiose dehydrogenase enzymes from each ofthe fungal species described herein can be targeted for geneticmodification. In some embodiments, the cellobiose dehydrogenase is froma fungal species in the family Chaetomiaceae. Some examples ofcellobiose dehydrogenase enzymes identified from Chaetomiaceae familymembers are set forth in Table 1, below. In some embodiments, thecellobiose dehydrogenase is from a fungal species selected fromSporotrichum cellulophilum, Thielavia heterothallica, Corynascusheterothallicus, Thielavia terrestris, Chaetomium globosum andMyceliophthora thermophila. Some cellobiose dehydrogenase enzymesidentified from these species are set forth in the table below. Theproteins listed in the table below are examples of cellobiosedehydrogenase that are known in the art, or identified herein as being acellobiose dehydrogenase.

TABLE 1 Cellobiose Dehydrogenase Sequences GMC oxred N GMC oxred CAccession Number Organism Domain Domain AAC26221 Myceliophthorathermophila 251-554 645-781 XP_001229896.1 Chaetomium globosum CBS226-529 620-757 148.51 JGIThite5441 Thielavia terrestris 253-555 647-783JGIThite4524 Thielavia terrestris  36-337 NA XP_001225932.1 Chaetomiumglobosum CBS  36-338 NA 148.51 JGIThite6738 Thielavia terrestris 249-550642-779 CDH2 derived from a C1 strain Myceliophthora thermophila 249-550NA XP_001226549.1 Chaetomium globosum CBS 249-521 549-667 148.51*Accession numbers for Thielavia terrestris refer to the U.S. Departmentof Energy (DOE) Joint Genome Institute (JGI) genome sequence

Certain amino acid sequences encoding cellobiose dehydrogenase areprovided herein. For example, the nucleotide sequence encodingMyceliophthora thermophila CDH1 is set forth herein as SEQ ID NO:1, andthe encoded amino acid sequence of Myceliophthora thermophila CDH1 isset forth as SEQ ID NO:2.

In some embodiments, the cellobiose dehydrogenase is cellobiosedehydrogenase EC 1.1.99.18. In some embodiments, the cellobiosedehydrogenase is a cellobiose dehydrogenase with the amino acid sequenceof Myceliophthora thermophila CDH1 as set forth in SEQ ID NO:2. In someother embodiments, the cellobiose dehydrogenase comprises an amino acidsequence provided in the GenBank entry of any one of the accessionnumbers set forth in Table 1. In some embodiments, the cellobiosedehydrogenase is encoded by a nucleic acid sequence that is at leastabout 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%,about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100% identical to SEQ ID NO:1. In some embodiments,the cellobiose dehydrogenase is encoded by a nucleic acid sequence thatis at least about 60%, about 61%, about 62%, about 63%, about 64%, about65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%,about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or about 100% identical to a nucleic acid sequenceencoding the amino acid sequence set forth as SEQ ID NO:2, or an aminoacid sequence provided in the GenBank entry of any one of the accessionnumbers set forth in Table 1. In some embodiments, the cellobiosedehydrogenase is encoded by a nucleic acid sequence that can selectivelyhybridize to SEQ ID NO:1, under moderately stringent or stringentconditions, as described hereinabove. In some embodiments, thecellobiose dehydrogenase is encoded by a nucleic acid sequence that canselectively hybridize under moderately stringent or stringent conditionsto a nucleic acid sequence that encodes SEQ ID NO:2, or an amino acidsequence provided in the GenBank entry of any one of the accessionnumbers set forth in Table 1.

In some embodiments, the cellobiose dehydrogenase comprises an aminoacid sequence with at least about 50%, about 51%, about 52%, about 53%,about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%,about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%,about 80%, about 81%, about 82%, about 83%, about 84%, about 85% about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100% similarity to the amino acid sequence set forth asSEQ ID NO:2, or an amino acid sequence provided in the GenBank entry ofany one of the accession numbers set forth in Table 1. Cellobiosedehydrogenase sequences can be identified by any of a variety of methodsknown in the art. For example, a sequence alignment can be conductedagainst a database, for example against the NCBI database, and sequenceswith the lowest HMM E-value can be selected.

In some embodiments, the fungal cells of the present invention have beengenetically modified to reduce the amount of cellobiose dehydrogenaseactivity from two or more endogenous cellobiose dehydrogenase enzymessecreted by the cell. In some embodiments, a first of the two or morecellobiose dehydrogenases comprises an amino acid sequence that is atleast about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, or about 99% identical to SEQ ID NO:2, and a second of the two ormore cellobiose dehydrogenase enzymes comprises an amino acid sequencethat is at least about 60%, about 61%, about 62%, about 63%, about 64%,about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%,about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to SEQ ID NO: 2.

Enzyme Mixtures

Also provided herein are enzyme mixtures that comprise at least one ormore cellulose hydrolyzing enzymes expressed by a fungal cell that hasbeen genetically modified to reduce the amount of endogenous cellobiosedehydrogenase activity secreted by the cell, as described herein.Cellulase enzymes are produced by a wide variety of microorganisms.Cellulases (and hemicellulases) from filamentous fungi and some bacteriaare widely exploited for many industrial applications that involveprocessing of natural fibers to sugars. It is contemplated that mixturesof any enzymes set forth herein will find use in the present invention.

In some embodiments, the enzyme mixture comprises at least one or morecellulose hydrolyzing enzymes expressed by a fungal cell that has beengenetically modified to reduce the amount of endogenous cellobiosedehydrogenase activity that is secreted by the cell, as describedherein. In some embodiments, the fungal cell is alignocellulose-utilizing cell from the family Chaetomiaceae. In someembodiments, the genetically modified fungal cell provided herein is aChaetomiaceae family member selected from Myceliophthora, Thielavia,Corynascus, or Chaetomium. In some other embodiments, the geneticallymodified fungal cell can also be an anamorph or teleomorph of aChaetomiaceae family member selected from Myceliophthora, Thielavia,Corynascus, or Chaetomium. In addition, the genetically modified fungalcell can also be selected from Sporotrichum or Acremonium orTalaromyces. It is also contemplated that the genetically modifiedfungal cell be selected from Ctenomyces, Thermoascus, and Scytalidium,including anamorphs and teleomorphs of fungal cells from those genera.In some embodiments, the fungal cell is a species selected fromSporotrichum cellulophilum, Thielavia heterothallica, Corynascusheterothallicus, Thielavia terrestris, Chaetomium globosum Talaromycesstipitatus and Myceliophthora thermophila, including anamorphs andteleomorphs thereof.

In addition to the enzymes described above, other enzymes such aslaccases find use in the mixtures of the present invention. Laccases arecopper containing oxidase enzymes that are found in many plants, fungiand microorganisms. Laccases are enzymatically active on phenols andsimilar molecules and perform a one electron oxidation. Laccases can bepolymeric and the enzymatically active form can be a dimer or trimer.

Mn-dependent peroxidases also find use in the mixtures of the presentinvention. The enzymatic activity of Mn-dependent peroxidase (MnP) in isdependent on Mn²⁺. Without being bound by theory, it has been suggestedthat the main role of this enzyme is to oxidize Mn²⁺ to Mn³⁺ (See e.g.,Glenn et al. Arch. Biochem. Biophys., 251:688-696 [1986]). Subsequently,phenolic substrates are oxidized by the Mn³⁺ generated.

Lignin peroxidases also find use in the mixtures of the presentinvention. Lignin peroxidase is an extracellular heme that catalyzes theoxidative depolymerization of dilute solutions of polymeric lignin invitro. Some of the substrates of LiP, most notably 3,4-dimethoxybenzylalcohol (veratryl alcohol, VA), are active redox compounds that havebeen shown to act as redox mediators. VA is a secondary metaboliteproduced at the same time as LiP by ligninolytic cultures of P.chrysosporium and without being bound by theory, has been proposed tofunction as a physiological redox mediator in the LiP-catalysedoxidation of lignin in vivo (See e.g., Harvey et al., FEBS Lett.,195:242-246 [1986]).

In some embodiments, it may be advantageous to utilize an enzyme mixturethat is cell-free. A cell-free enzyme mixture typically comprisesenzymes that have been separated from any cells, including the cellsthat secreted the enzymes. Cell-free enzyme mixtures can be preparedusing any of a variety of suitable methodologies that are known in theart (e.g., filtration or centrifugation). In some embodiments, theenzyme mixture is partially cell-free, substantially cell-free, orentirely cell-free.

In some embodiments, two or more cellulases and any additional enzymespresent in the cellulase enzyme mixture are secreted from a singlegenetically modified fungal cell or by different microbes in combined orseparate fermentations. Similarly, two or more cellulases and anyadditional enzymes present in the cellulase enzyme mixture may beexpressed individually or in sub-groups from different strains ofdifferent organisms and the enzymes combined in vitro to make thecellulase enzyme mixture. It is also contemplated that the cellulasesand any additional enzymes in the enzyme mixture are expressedindividually or in sub-groups from different strains of a singleorganism, and the enzymes combined to make the cellulase enzyme mixture.

In some embodiments, the enzyme mixture comprises at least one or morecellulose hydrolyzing enzymes expressed by a fungal cell that has beengenetically modified to reduce the amount of endogenous cellobiosedehydrogenase activity that is secreted by the cell, as describedherein. In some embodiments, the fungal cell is alignocellulose-utilizing cell from the family Chaetomiaceae. In someembodiments, the genetically modified fungal cell provided herein is aChaetomiaceae family member selected from Myceliophthora, Thielavia,Corynascus, and Chaetomium. The genetically modified fungal cell canalso be an anamorph or teleomorph of a Chaetomiaceae family memberselected from Myceliophthora, Thielavia, Corynascus, and Chaetomium. Inaddition, the genetically modified fungal cell can also be selected fromSporotrichum, Acremonium, Ctenomyces, Scytalidium and Thermoascus,including anamorphs and teleomorphs of fungal cells from these genera.In some embodiments, the fungal cell is a species selected fromSporotrichum cellulophilum, Thielavia heterothallica, Corynascusheterothallicus, Thielavia terrestris, Chaetomium globosum, Talaromycesstipitatus, and Myceliophthora thermophila, including anamorphs andteleomorphs thereof.

In some embodiments, the cellulase enzyme mixture of the presentinvention is produced in a fermentation process in which the fungalcells described herein are grown in submerged liquid culturefermentation. In some embodiments, submerged liquid fermentations offungal cells are incubated using batch, fed-batch or continuousprocessing. In a batch process, all the necessary materials, with theexception of oxygen for aerobic processes, are placed in a reactor atthe start of the operation and the fermentation is allowed to proceeduntil completion, at which point the product is harvested. In someembodiments, batch processes for producing the enzyme mixture of thepresent invention are carried out in a shake-flask or a bioreactor. Insome embodiments in which a fed-batch process is used, the culture isfed continuously or sequentially with one or more media componentswithout the removal of the culture fluid. In continuous processes, freshmedium is supplied and culture fluid is removed continuously atvolumetrically equal rates to maintain the culture at a steady growthrate. Those of skill in the art will appreciate that fermentation mediumis typically liquid, and comprises a carbon source, a nitrogen source aswell as other nutrients, vitamins and minerals which can be added to thefermentation media to improve growth and enzyme production of the fungalcells. These other media components may be added prior to,simultaneously with or after inoculation of the culture with the fungalcells.

In some embodiments of the process for producing the enzyme mixture ofthe present invention, the carbon source comprises a carbohydrate thatwill induce the expression of the cellulase enzymes from the fungalcell. For example, in some embodiments, the carbon source comprises oneor more of cellulose, cellobiose, sophorose, xylan, xylose, xylobiose,and/or related oligo- or poly-saccharides known to induce expression ofcellulases and beta-glucosidase in such fungal cells. In someembodiments utilizing batch fermentation, the carbon source is added tothe fermentation medium prior to or simultaneously with inoculation. Insome embodiments utilizing fed-batch or continuous operations, thecarbon source is supplied continuously or intermittently during thefermentation process. For example, in some embodiments, the carbonsource is supplied at a carbon feed rate of between about 0.2 and about2.5 g carbon/L of culture/h, or any suitable amount therebetween.

The methods for producing the enzyme mixture of the present inventionmay be carried at any suitable temperature, typically from about 20° C.to about 100° C., or any suitable temperature therebetween, for examplefrom about 20 ° C. to about 80° C. , 25° C. to about 65° C., or anysuitable temperature therebetween, or from about 20° C., about 22° C.,about 25° C., about 26° C., about 27° C., about 28° C., about 29° C.,about 30° C., about 32° C., about 35° C., about 37° C., about 40° C.,about 45° C., about 50° C., about 55° C., about 60° C., about 65° C.,about 70° C., about 75° C., about 80° C,about 85° C. C, about 90° C.,about 95° C., and/or any suitable temperature therebetween.

The methods for producing enzyme mixture of the present invention may becarried out at any suitable pH, typically from about 3.0 to 8.0, or anysuitable pH therebetween, for example from about pH 3.5 to pH 6.8, orany suitable pH therebetween, for example from about pH 3.0, about 3.2,about 3.4, about 3.5, about 3.7, about 3.8, about 4.0, about 4.1, about4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8,about 4.9, about 5.0, about 5.2, about 5.4, about 5.5, about 5.7, about5.8, about 6.0, about 6.2, about 6.5, about 6.8, about 7.0, about 7.2,about 7.5, about 8.0, or any suitable pH therebetween.

In some embodiments, the enzyme mixture is contained in a vesselcomprising a genetically modified fungal cell as described herein. Insome embodiments, the vessel comprises a liquid medium. In someembodiments, the vessel is a flask, bioprocess reactor, or any othersuitable container. In some embodiments, the enzyme mixture is in aliquid volume. In some embodiments, the liquid volume can be greaterthan about 0.01 mL, about 0.1 mL, about 1 mL, about 10 mL, about 100 mL,about 1000 mL, or greater than about 10 L, about 50 L, about 100 L,about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about700 L, about 800 L, about 900 L, about 1000 L, about 10,000 L, about50,000 L, about 100,000 L, about 250,000 L, about 500,000 L or greaterthan about 1,000,000 L.

In some embodiments, following fermentation, the fermentation mediumcontaining the fungal cells is used, or the fermentation mediumcontaining the fungal cells and the enzyme mixture is used, or theenzyme mixture is separated from the fungal cells, for example byfiltration or centrifugation, and the enzyme mixture in the fermentationmedium is used. In some embodiments, low molecular solutes such asunconsumed components of the fermentation medium are removed byultrafiltration. In some embodiments, the enzyme mixture is concentratedby evaporation, precipitation, sedimentation, filtration, or anysuitable means. In some embodiments, chemicals such as glycerol,sucrose, sorbitol, etc., are added to stabilize the enzyme mixture. Insome embodiments, other chemicals, such as sodium benzoate or potassiumsorbate, are added to the enzyme mixture to prevent growth of microbialcontaminants.

Methods for Generating Glucose

The present invention also provides processes for generating glucose,comprising contacting cellulose with the enzyme mixture describedherein. For example, in some embodiments, the process comprisescontacting cellulose with an enzyme mixture comprising two or morecellulose hydrolyzing enzymes, wherein at least one of the two or morecellulose hydrolyzing enzymes is expressed by a fungal cell as describedherein. In some embodiments, the method for generating glucose fromcellulose using the enzyme mixture is batch hydrolysis, continuoushydrolysis, or a combination thereof. In some embodiments, thehydrolysis is agitated, unmixed, or a combination thereof.

The methods for generating glucose from cellulose may be carried out atany suitable temperature, including between about 30° C. and about 80°C., or any suitable temperature therebetween, for example a temperatureof about 30° C., about 35° C., about 40° C., about 45° C., about 50° C.,about 55° C., about 60° C., about 65° C., about 70° C., about 75° C.,about 80° C. or any suitable temperature therebetween, and a pH of about3.0 to about 8.0, or any suitable pH therebetween, for example at a pHof about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5,about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or any sutiablepH therebetween. The initial concentration of cellulose in thehydrolysis reactor, prior to the start of hydrolysis, is preferablyabout 0.1% (w/w) to about 15% (w/w), or any suitable amounttherebetween, for example about 2, about 4, about 6, about 8, about 10,about 12, about 14, about 15%, or any suitable amount therebetween.

The dosage of the cellulase enzyme mixture may be about 0.1 to about 100mg protein per gram cellulose, or any suitable amount therebetween, forexample about 0.1, about 0.5, about 1, about 5, about 10, about 15,about 20, about 25, about 30, about 40, about 50, about 60, about 70,about 80, about 90, about 100 mg protein per gram cellulose or anysuitable amount therebetween. The hydrolysis may be carried out for atime period of about 0.5 hours to about 200 hours, or any suitable timetherebetwee. For example, in some embodiments, the hydrolysis is carriedout for a period of about 15 hours to about 100 hours, or any timetherebetween, or it may be carried out for about 0.5 hour, about 1 hour,about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 15hours, about 20 hours, about 25 hours, about 30 hours, about 35 hours,about 40 hours, about 45 hours, about 50 hours, about 55 hours, about 60hours, about 65 hours, about 70 hours, about 75 hours, about 80 hours,about 85 hours, about 90 hours, about 95 hours, about 100 hours, about120 hours, about 140 hours, about 160 hours, about 180 hours, about 200hours, or any suitable time therebetween. It should be appreciated thatthe reaction conditions are not meant to limit the invention in anymanner and may be adjusted as desired by those of skill in the art.

In some embodiments, the enzymatic hydrolysis is typically carried outin a hydrolysis reactor. The enzyme mixture is added to the pretreatedlignocellulosic feedstock (also referred to as the “substrate”) priorto, during, or after the addition of the substrate to the hydrolysisreactor.

In methods of contacting cellulosic material with an enzyme mixture,various environmental conditions may be adjusted according to anyvariety of methods known in the art in order to maximize the formationof a hydrolysis product such as glucose. For example, temperature, pH, %dissolved oxygen, and stirring speed can each be independently adjusted.In some embodiments, the enzyme mixture is a cell-free mixture, asdescribed herein.

The methods for generating glucose, as described herein, using theenzyme mixture with reduced cellobiose dehydrogenase activity result ina higher yield of glucose from the enzymatically hydrolyzed cellulosethan a corresponding process using an enzyme mixture with its fullcomplement of cellobiose dehydrogenase activity. Further, such methodsresult in decreased conversion of the cellobiose products in theenzymatic hydrolysate to oxidized products.

In some embodiments of the methods using the genetically modified cellsand/or enzyme mixtures provided herein, improved glucose yield can bemeasured and quantified. As described herein, glucose yield can bedescribed in terms of the amount of generated glucose per theoreticalmaximum glucose yield, or in terms of Gmax. It will be appreciated bythose skilled in the art that when calculating theoretical values suchas Gmax and theoretical maximum glucose yield, the mass of two hydrogenatoms and one oxygen atom that are added to the glucose molecule in thecourse of the hydrolysis reaction is taken into account. For example,when a polymer of “n” glucose units is hydrolyzed, (n−1) units of waterare added to the glucose molecules formed in the hydrolysis, so theweight of the glucose produced is about 10% greater than the weight ofcellulose consumed in the hydrolysis (e.g., hydrolysis of 1 g celluloseproduces about 1.1 g glucose). Thus, as an example, where 5 g of totalavailable cellulose is present at the beginning of a hydrolysisreaction, and 2 g of residual cellulose remains after the reaction, thetotal hydrolyzed cellulose is 3 g cellulose. A theoretical maximumglucose yield of 100% (w/w) under the reaction conditions is about 5.5 gof glucose. Gmax is calculated based on the 3 g of cellulose that wasreleased or converted in the reaction by hydrolysis. Thus, in thisexample, a Gmax of 100% (w/w) is about 3.3 g of glucose. Celluloselevels, either the total available amount present in the substrate orthe amount of unhydrolyzed or residual cellulose, can be quantified byany of a variety of suitable methods known in the art, such as by IRspectroscopy or by measuring the amount of glucose generated byconcentrated acid hydrolysis of the cellulose (See e.g., U.S. Pat. Nos.6,090,595 and 7,419,809, both of which are incorporated by referenceherein in their entireties).

For example, in some embodiments, the cellulose content is determined byacid hydrolysis of the cellulose, followed by glucose concentrationdetermination, taking into account the water necessary to hydrolyze thecellulose (See e.g., U.S. Pat. Nos. 6,090,595 and 7,419,809). In oneexample, a slurry of feedstock is centrifuged, washed with water, andsuspended in sulfuric acid at a net sulfuric acid concentration of 70%.The slurry is incubated at 40° C. for 30 minutes, followed by dilutionin deionized water to 2% sulfuric acid. At this time point, the samplesare steam-autoclaved at 121° C. for 1 hour, to convert the oligomers tomonomeric glucose. The glucose concentration is measured by HPLC or anysuitable enzymatic assay. In some alternative embodiments, the cellulosecontent is analyzed by infrared spectroscopy as described in Example 1.For example, solids can be washed and placed on the detection crystal ofan infrared spectrometer and the absorbance measured between 500-4000cm⁻¹.

Glucose levels can be quantified by any of a variety of suitable methodsknown in the art (See e.g., U.S. Pat. Nos. 6,090,595 and 7,419,809). Forexample, glucose concentrations can be determined using a coupledenzymatic assay based on glucose oxidase and horseradish peroxidase (Seee.g., Trinder, Ann. Clin. Biochem., 6:24-27 [1969], which isincorporated herein by reference in its entirety). Additional methods ofglucose quantification include chromatographic methods (See e.g., U.S.Pat. Nos. 6,090,595 and 7,419,809). Cellobiose levels can be measured byany number of suitable HPLC methods known to those of skill in the art(See e.g., Kotiranta et al., Appl. Biochem. Biotechnol., 81:81-90[1999]), which is incorporated herein by reference in its entirety).

Similarly, decreased conversion of cellobiose and glucose products tooxidized products such as cellobionolactone and gluconolactone can bequantified by any of a variety of suitable methods known in the art. Forexample, products of glucose or cellobiose oxidation can be detected andquantified using infrared spectroscopy, or by chromatographicmethodologies such as HPLC (See e.g., Rakotomanga et al., J. Chromatog.B., 4:277-284 [1991]; and Mansfield et al., Appl. Environ. Microbiol.,64:3804-3809 [1997], both of which are incorporated herein by referencein their entireties). Accordingly, total oxidation of glucose orcellobiose can be determined, for example, as a function of totaloxidation products per theoretical maximum glucose yield, or as afunction of Gmax.

Cellulosic Material

Any material containing cellulose finds use in the present invention.The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemicellulose, and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened by polymericlignin covalently cross-linked to hemicellulose. Cellulose is ahomopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan,while hemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees.Cellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (See e.g., Wiselogel et al., inCharles E. Wyman, (ed.), Handbook on Bioethanol, Taylor & Francis,Washington D.C. [1995], at pp. 105-118; Wyman, Biores. Technol., 50:3-16[1994]; Lynd, Appl. Biochem. Biotechnol., 24/25: 695-719 [1990]; andMosier et al., Adv. Biochem. Eng. Biotechnol., 65:23-40 [1999]). It isunderstood that in some embodiments, the cellulose is in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In some embodiments, the cellulosicmaterial is lignocellulose.

A pretreated lignocellulosic feedstock is a material of plant originthat, prior to pretreatment, contains at least 10% cellulose (dryweight), more preferably greater than about 30% cellulose, even morepreferably greater than 40% cellulose, for example about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%,about 38%, about 39%, about 40%, about 41%. about 42%, about 43%, about44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, or any suitable percent therebetween, and atleast about 10% lignin (dry weight), or at least about 12% (dry weight)and that has been subjected to physical and/or chemical processes tomake the fiber more accessible and/or receptive to the actions ofcellulolytic enzymes. In some embodiments, the lignocellulosic feedstockmay contain higher levels of cellulose after pretreatment. For example,if acid pretreatment is employed, the hemicellulose component ishydrolyzed, which increases the relative level of cellulose. In thiscase, the pretreated feedstock may contain greater than about 20%cellulose and greater than about 12% lignin.

Lignocellulosic feedstocks that find use in the invention include, butare not limited to, agricultural residues such as corn stover, wheatstraw, barley straw, rice straw, oat straw, canola straw, sugarcanestraw and soybean stover; fiber process residues such as corn fiber,sugar beet pulp, pulp mill fines and rejects or sugar cane bagasse;forestry residues such as aspen wood, other hardwoods, softwood, andsawdust; or grasses such as switch grass, miscanthus, cord grass, andreed canary grass. In some embodiments, the lignocellulosic feedstock isfirst subjected to size reduction by any of a variety of methodsincluding, but not limited to, milling, grinding, agitation, shredding,compression/expansion, and/or other types of mechanical action. Sizereduction by mechanical action can be performed by any type of equipmentadapted for the purpose, for example, but not limited to, a hammer mill

Pretreatment

In some embodiments, a substrate of the enzyme mixture comprisespretreated cellulosic material. Thus, for example, in some methodsdescribed herein, any pretreatment process known in the art can be usedto disrupt plant cell wall components of cellulosic material (See e.g.,Chandra et al., Adv. Biochem. Engin. Biotechnol., 108: 67-93 [2007];Galbe and Zacchi, Adv. Biochem. Engin. Biotechnol., 108: 41-65 [2007];Hendriks and Zeeman, Biores. Technol., 100: 10-18 [2009]; Mosier et al.,Biores. Technol., 96: 673-686 [2005]; Taherzadeh and Karimi, Int. J.Mol. Sci., 9:1621-1651 [2008]; and Yang and Wyman, Biofuels Bioprod.Bioref. Biofpr. 2: 26-40 [2008]; all of which are hereby incorporated byreference in their entireties).

In some embodiments, the cellulosic material is subjected to particlesize reduction, pre-soaking, wetting, washing, or conditioning prior topretreatment using any of a variety of suitable methods known in theart. Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber expansion, dilute ammoniapretreatment, organosolv pretreatment, and biological pretreatment.Additional pretreatments include ammonia percolation, ultrasound,electroporation, microwave, supercritical CO₂, supercritical H₂O, ozone,and gamma irradiation pretreatments. In some embodiments, the cellulosicmaterial is pretreated before hydrolysis and/or fermentation. In someembodiments, pretreatment is preferably performed prior to thehydrolysis. In some alternative embodiments, pretreatment is carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In some embodiments, thepretreatment step itself results in some conversion of biomass tofermentable sugars, even in absence of enzymes.

Steam Pretreatment. In steam pretreatment, cellulosic material is heatedto disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions(e.g., hemicelluloses), accessible to enzymes. Cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. In some embodiments,steam pretreatment is preferably done at about 140° C. to about 230° C.,while in other embodiments it is done at about 160° C. to about 200° C.,and in additional embodiments, it is done at about 170° C. to about 190°C., where the optimal temperature range depends on any addition of achemical catalyst. In some embodiments, residence time for the steampretreatment is about 1 to about 15 minutes, while in other embodimentsit is about 3 to about 12 minutes, and in still other embodiments, it isabout 4 to about 10 minutes, where the optimal residence time depends ontemperature range and any addition of a chemical catalyst. Steampretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment.Steam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Seee.g., U.S. Pat. No. 4,451,648; Duff and Murray, Biores. Technol.,855:1-33 [1996]; Galbe and Zacchi, Appl. Microbiol. Biotechnol., 59:618-628 [2002]; and U.S. Patent Appln. Publ. No. 2002/0164730, all ofwhich are incorporated herein by reference in their entireties). Duringsteam pretreatment, hemicellulose acetyl groups are cleaved and theresulting acid autocatalyzes partial hydrolysis of the hemicellulose tomonosaccharides and oligosaccharides. Lignin is removed to only alimited extent. A catalyst such as H₂SO₄ or SO₂ (typically about 0.3 toabout 3% w/w) is often added prior to steam pretreatment, whichdecreases the time and temperature, increases the recovery, and improvesenzymatic hydrolysis (See e.g., Ballesteros et al., Appl. Biochem.Biotechnol., 129-132: 496-508 [2006]; Varga et al., Appl. Biochem.Biotechnol., 113-116: 509-523 [2004]; and Sassner et al., Enz. Microb.Technol., 39: 756-762 [2006]).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, but are not limited to, dilute acidpretreatment, dilute alkali pretreatment (See e.g., U.S. Pat. Appln.Pub. Nos. 2007/0031918 and 2007/0037259), lime pretreatment, wetoxidation, ammonia fiber/freeze explosion or expansion (AFEX), ammoniapercolation (APR), dilute ammonia pretreatment, and organosolvpretreatments (See e.g., WO 2006/110891, WO 2006/11899, WO 2006/11900,and WO 2006/110901).

In dilute acid pretreatment, cellulosic material is mixed with diluteacid, typically H₂SO₄, and water to form a slurry, heated by steam tothe desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs (e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors) (Seee.g., Duff and Murray, supra; Schell et al., Biores. Technol., 91:179-188 [2004]; and Lee et al., Adv. Biochem. Eng. Biotechnol., 65:93-115 [1999]).

In some embodiments, lime pretreatment is performed with calciumcarbonate, sodium hydroxide, or ammonia at low temperatures of about 85°C. to about 150° C. and residence times from about 1 hour to severaldays (Wyman et al., Biores. Technol., 96: 1959-1966 [2005]; and Mosieret al., Biores. Technol. 96: 673-686 [2005]).

Wet oxidation is a thermal pretreatment performed typically at about180° C. to about 200° C. for about 5 to about 15 minutes with additionof an oxidative agent such as hydrogen peroxide or over-pressure ofoxygen (See e.g., Schmidt and Thomsen, Biores. Technol., 64:139-151[1998]; Palonen et al., Appl. Biochem. Biotechnol., 117: 1-17 [2004];Varga et al., Biotechnol. Bioeng., 88: 567-574 [2004]; Martin et al., J.Chem. Technol. Biotechnol., 81: 1669-1677 [2006]). The pretreatment isperformed at preferably about 1% to about 40% dry matter, about 2 toabout 30% dry matter, or about 5 to about 20% dry matter, and often theinitial pH is increased by the addition of an alkali such as sodiumcarbonate. In some embodiments, a modification of the wet oxidationpretreatment method, known as wet explosion (combination of wetoxidation and steam explosion), finds use. This method can handle drymatter up to about 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (Seee.g., WO 2006/032282).

In some embodiments, ammonia fiber expansion (AFEX) finds use. Thismethod involves treating cellulosic material with liquid or gaseousammonia at moderate temperatures such as about 90 to about 100° C. andhigh pressure such as about 17 to about 20 bar for about 5 to about 10minutes, where the dry matter content can be as high as about 60% (Seee.g., Gollapalli et al., Appl. Biochem. Biotechnol., 98: 23-35 [2002];Chundawat et al., Biotechnol. Bioeng., 96:219-231 [2007]; Alizadeh etal., Appl. Biochem. Biotechnol., 121: 1133-1141 [2005]; and Teymouri etal., Biores. Technol., 96: 2014-2018 [2005]). AFEX pretreatment resultsin the depolymerization of cellulose and partial hydrolysis ofhemicellulose. Lignin-carbohydrate complexes are cleaved. Dilute ammoniapretreatment utilizes more dilute solutions of ammonia than AFEX and maybe conducted at a temperature of about 100° C. to about 150° C., or anysuitable temperature therebetween (See e.g., U.S. Pat. Appln. Pub. Nos.2007/0031918 and 2007/0037259, herein incorporated by reference in theirentireties). In some embodiments, the duration of the dilute ammoniapretreatment is about 1 to about 20 minutes, or any suitable durationtherebetween.

In some embodiments, organosolv pretreatment finds use. This methoddelignifies cellulosic material by extraction using aqueous ethanol(about 40% to about 60% ethanol) at about 160° C. to about 200° C. forabout 30 to about 60 minutes (See e.g., Pan et al., Biotechnol. Bioeng.,90: 473-481 [2005]; Pan et al., Biotechnol. Bioeng., 94: 851-861 [2006];and Kurabi et al., Appl. Biochem. Biotechnol., 121: 219-230 [2005]).Sulfuric acid is usually added as a catalyst. In organosolvpretreatment, the majority of hemicellulose is removed.

Other examples of suitable pretreatment methods are known in the art(See e.g., Schell et al., Appl. Biochem. Biotechnol., 105:69-85 [2003];Mosier et al., Biores. Technol., 96: 673-686 [2005]; and U.S. Pat.Appln. Publ. No. 2002/0164730).

In some embodiments, the chemical pretreatment is preferably carried outas an acid treatment, and more preferably as a continuous dilute and/ormild acid treatment. The acid is typically sulfuric acid, but otheracids can also be used, such as nitric acid, phosphoric acid, hydrogenchloride or mixtures thereof. Mild acid treatment is conducted in the pHrange of about 1 to about 5, or about 1 to about 4, or about 1 to about3. In some embodiments, the acid concentration is in the range of fromabout 0.01 to about 20 wt % acid, while in other embodiments, it is inthe range of from about 0.05 to about 10 wt % acid, in otherembodiments, it is in the range of from about 0.1 to about 5 wt % acid,and in still other embodiments, it is in the range of from about 0.2 toabout 2.0 wt % acid. The acid is contacted with cellulosic material andheld at a temperature in the range of preferably about 160° C. to about220° C., and more preferably about 165° C. to about 195° C., for periodsranging from seconds to minutes to (e.g., about 1 second to about 60minutes).

In some embodiments, pretreatment takes place in an aqueous slurry. Insome embodiments, cellulosic material is present during pretreatment inamounts preferably between about 10 to about 80 wt %, or about 20 toabout 70 wt %, or about 30 to about 60 wt %, or about 50 wt %. Thepretreated cellulosic material can be unwashed or washed using anysuitable method known in the art (e.g., washed with water).

Physical Pretreatment. Physical pretreatment can involve high pressureand/or high temperature (steam explosion). In some embodiments, highpressure physical pretreatment involves pressure in the range of about300 to about 600 psi, or about 350 to about 550 psi, or about 400 toabout 500 psi, or about 450 psi. In some other embodiments, hightemperature pretreatment involves the use of treatment temperatures inthe range of about 100° C. to about 300° C., or about 140° C. to about235° C. In some embodiments, mechanical pretreatment is performed in abatch-process, steam gun hydrolyzer system that uses high pressure andhigh temperature as defined above (e.g., Sunds Hydrolyzer; SundsDefibrator AB, Sweden).

Combined Physical and Chemical Pretreatment. In some embodiments,combined physical and chemical pretreatments find use. Indeed,cellulosic material can be pretreated both physically and chemically.For example, in some embodiments, the pretreatment step involves diluteor mild acid treatment and high temperature and/or pressure treatment.The physical and chemical pretreatments can be carried out sequentiallyor simultaneously, as desired. In some additional embodiments,mechanical pretreatment is also used in conjunction with the physicaland chemical pretreatments. Thus, in some embodiments, cellulosicmaterial is subjected to mechanical, chemical, or physical pretreatment,or any combination thereof, to promote the separation and/or release ofcellulose, hemicellulose, and/or lignin.

Biological Pretreatment. In some embodiments, biological pretreatmenttechniques find use. In some embodiments, these methods involve applyinglignin-solubilizing microorganisms (See e.g., Hsu, in Wyman (ed.),Handbook on Bioethanol: Production and Utilization, Taylor & Francis,Washington, D.C., at pp. 179-212 [1996]; Ghosh and Singh, Adv. Appl.Microbiol., 39:295-333 [1993]; McMillan, in Baker and Overend (eds.),Enzymatic Conversion of Biomass for Fuels Production, ACS SymposiumSeries 566, American Chemical Society, Washington, D.C., chapter 15[1994]; Gong et al., Adv. Biochem. Engineer. Biotechnol., 65: 207-241[1999]; Olsson and Hahn-Hagerdal, Enz. Microb. Tech., 18:312-331 [1996];and Vallander and Eriksson, Adv. Biochem. Eng. Biotechnol. 42: 63-95[1990]).

In some embodiments, the soluble compounds derived from pretreatmentprocess are subsequently separated from the solids. For example, in someembodiments, the separation step comprises one or more of standardmechanical means (e.g., screening, sieving, centrifugation orfiltration) to achieve the separation. In some other embodiments, thesoluble compounds are not separated from the solids followingpretreatment. Those of skill in the art appreciate that pretreatment maybe conducted as a batch, fed-batch or continuous process. It will alsobe appreciated that pretreatment may be conducted at low, medium or highsolids consistency as desired (See e.g., WO2010/022511, which isincorporated herein by reference in its entirety).

Fermentation

In some embodiments, methods for generating sugar(s) described hereinfurther comprise fermentation of the resultant sugar(s) to an endproduct. Fermentation involves the conversion of a sugar source to anend product through the use of a fermenting organism. Any suitableorganism finds use in the present invention, including bacterial andfungal organisms (e.g., yeast and filamentous fungi), suitable forproducing a desired end product. Especially suitable fermentingorganisms are able to ferment (i.e., convert), sugars, such as glucose,fructose, maltose, xylose, mannose and/or arabinose, directly orindirectly into a desired end product. Examples of fermenting organismsinclude fungal organisms such as yeast. In some embodiments, yeaststrains, including but not limited to the following genera find use: thegenus Saccharomyces (e.g., S. cerevisiae and S. uvarum); Pichia (e.g.,P.stipitis and P. pastoris); Candida (e.g.,C. utilis, C.arabinofermentans, C. diddensii, C. sonorensis, C. shehatae, C.tropicalis, and C. boidinii). Other fermenting organisms include, butare not limited to strains of Zymomonas, Hansenula (e.g., H. polymorphaand H. anomala), Kluyveromyces (e.g., K fragilis), andSchizosaccharomyces (e.g., S. pombe).

In some embodiments, the fermenting organisms are strains of Escherichia(e.g., E. coli), Zymomonas (e.g., Z. mobilis), Zymobacter (e.g., Z.palmae), Klebsiella (e.g., K. oxytoca), Leuconostoc (e.g., L.mesenteroides), Clostridium (e.g., C. butyricum), Enterobacter (e.g., E.aerogenes) and Thermoanaerobacter (e.g., Thermoanaerobacter BG1L1 [Seee.g., Georgieva and Ahring, Appl. Microbiol, Biotech., 77: 61-86] T.ethanolicus, T. thermosaccharolyticum, or T. mathranii), Lactobacillus,Corynebacterium glutamicum R, Bacillus thermoglucosidaisus, andGeobacillus thermoglucosidasius. It is not intended that the fermentingorganism be limited to these particular strains, as any suitableorganism finds use in the present invention.

The fermentation conditions depend on the desired fermentation productand can easily be determined by one of ordinary skill in the art. Insome embodiments involving ethanol fermentation by yeast, fermentationis typically ongoing for between about 1 hour to about 120 hours, orabout 12 to about 96 hours. In some embodiments, the fermentation iscarried out at a temperature between about 20° C. to about 40° C., orbetween about 26° C. and about 34° C., or about 32° C. In someembodiments, the fermentation pH is from about pH 3 to about pH 7, whilein some other embodiments, the pH is about 4 to about 6.

In some embodiments, enzymatic hydrolysis and fermentation are conductedin separate vessels, so that each biological reaction can occur underits respective optimal conditions (e.g., temperature). In some otherembodiments, the methods for producing glucose from cellulose areconducted simultaneously with fermentation in a simultaneoussaccharification and fermentation (i.e., “SSF”) reaction. In someembodiments, SSF is typically carried out at temperatures of about 28°C. to about 50° C., or about 30° C. to about 40° C., or about 35° C. toabout 38° C., which is a compromise between the about 50° C. optimum formost cellulase enzyme mixtures and the about 28° C. to about 30° C.optimum for most yeast. However, it is not intended that the presentinvention be limited to any particular temperature, as any suitabletemperature finds use in the present invention.

In some embodiments, the methods for generating glucose further comprisefermentation of the glucose to a desired end product. It is not intendedthat the methods provided herein be limited to the production of anyspecific end product. In some embodiments, end products include fuelalcohols or precursor industrial chemicals. For example, in someembodiments, fermentation products include precursor industrialchemicals such as alcohols (e.g., ethanol, methanol and/or butanol);organic acids (e.g., butyric acid, citric acid, acetic acid, itaconicacid, lactic acid, and/or gluconic acid); ketones (e.g., acetone); aminoacids (e.g., glutamic acid); gases (e.g., H₂ and/or CO₂); antimicrobials(e.g., penicillin and/or tetracycline); enzymes; vitamins (e.g.,riboflavin, B₁₂, and/or beta-carotene); and/or hormones. In someembodiments, the end product is a fuel alcohol. Suitable fuel alcoholsare known in the art and include, but are not limited to lower alcoholssuch as methanol, ethanol, butanol and propyl alcohols.

Increased Expression of Saccharide Hydrolyzing Enzymes

In some embodiments provided herein, the fungal cell is furthergenetically modified to increase its production of one or moresaccharide hydrolyzing enzymes. For example, in some embodiments, thefungal cell overexpresses a homologous or heterologous gene encoding asaccharide hydrolysis enzyme such as beta-glucosidase. In someembodiments, the one or more saccharide hydrolysis enzyme is a cellulaseenzyme described herein. For example, in some embodiments, the enzyme isany one of a variety of endoglucanases, cellobiohydrolases,beta-glucosidases, endoxylanases, beta-xylosidases,arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases,feruloyl esterases, and alpha-glucuronyl esterases, and/or any otherenzyme involved in saccharide hydrolysis. In some embodiments, thefungal cell is genetically modified to increase expression ofbeta-glucosidase. Thus, in some embodiments, the fungal cell comprises apolynucleotide sequence for increased expression ofbeta-glucosidase-encoding polynucleotide. In some embodiments, thefungal cell is further genetically modified to delete polynucleotidesencoding one or more endogenous cellobiose dehydrogenase enzymes.

In some embodiments, the saccharide hydrolyzing enzyme is endogenous tothe fungal cell, while in other embodiments, the saccharide hydrolyzingenzyme is exogenous to the fungal cell. In some additional embodiments,the enzyme mixture further comprises a saccharide hydrolyzing enzymethat is heterologous to the fungal cell. Still further, in someembodiments, the methods for generating glucose comprise contactingcellulose with an enzyme mixture that comprises a saccharide hydrolyzingenzyme that is heterologous to the fungal cell.

In some embodiments, a fungal cell is genetically modified to increasethe expression of a saccharide hydrolysis enzyme using any of a varietyof suitable methods known to those of skill in the art. In someembodiments, the hydrolyzing enzyme-encoding polynucleotide sequence isadapted for increased expression in a host fungal cell. As used herein,a polynucleotide sequence that has been adapted for expression is apolynucleotide sequence that has been inserted into an expression vectoror otherwise modified to contain regulatory elements necessary forexpression of the polynucleotide in the host cell, positioned in such amanner as to permit expression of the polynucleotide in the host cell.Such regulatory elements required for expression include promotersequences, transcription initiation sequences and, optionally, enhancersequences. For example, in some embodiments, a polynucleotide sequenceis inserted into a plasmid vector adapted for expression in the fungalhost cell.

EXPERIMENTAL

The present invention is described in further detail in the followingExamples, which are not in any way intended to limit the scope of theinvention as claimed.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and μg (micrograms); L and 1 (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) (hour(s)); U(units); MW (molecular weight); rpm (rotations per minute); ° C.(degrees Centigrade); wt % (weight percent); w.r.t. (with regard to);DNA (deoxyribonucleic acid); RNA (ribonucleic acid); HPLC (high pressureliquid chromatography); MS (mass spectroscopy); LC (liquidchromatography); LC/MS (liquid chromatography/mass spectroscopy);LC/MS/MS (liquid chromatography/multi-stage mass spectroscopy); HMF(hydroxymethylfurfural); YPD (Yeast extract 10 g/L; Peptone 20 g/L;Dextrose 20 g/L); DCPIP (2,6-dichlorophenolindophenol); CV (columnvolume); NREL (National Renewable Energy Laboratory, Golden, Colo.); ARS(ARS Culture Collection or NRRL Culture Collection, Peoria, Ill.);Lallemand (Lallemand Ethanol Technology, Milwaukee, Wis.); Cayla(Cayla-InvivoGen, Toulouse, France); Agilent New Brunswick (NewBrunswick Scientific Co., Edison, N.J.); Sigma (Sigma Aldrich, St.Louis, Mo.); Eppendorf (Eppendorf AG, Hamburg, Germany); GE Healthcare(GE Healthcare, Waukesha, Wis.); Bruker Optics (Bruker Optics, Inc.,Billerica, Mass.); Specac (Specac, Inc., Cranston, R.I.); Invitrogen(Invitrogen, Corp., Carlsbad, Calif.); Alphalyse (Alphalyse, Inc., PaloAlto, Calif.); Promega (Promega, Corp., Madison, Wis.); Sartorius(Sartorius-Stedim Biotech, SA, Aubagne, France); Finnzymes (FinnzymesOy, Espoo, FI [part of Thermo Fisher Scientific]), CalBiochem(CalBiochem, EMD Chemicals, Inc., Gibbstown, N.J.); and Bio-Rad (Bio-RadLaboratories, Hercules, Calif.).

The following CDH sequences from M. thermophila (C1) find use in thepresent invention. SEQ ID NOS:1 and 2 provide CDH1 nucleic acid andamino acid sequences, respectively. SEQ ID NO:3 is the amino acidsequence of CDH2, while SEQ ID NO:4 is the amino acid sequence of CDH3,SEQ ID NO:5 is the amino acid sequence of CDH4, SEQ ID NO:6 is the aminoacid sequence of CDH5, SEQ ID NO:7 is the amino acid sequence of CDH6,and SEQ ID NO:8 is the amino acid sequence of CDH7.

CDH1: (SEQ ID NO: 1) atgaggacctcctctcgtttaatcggtgcccttgcggcggcactcttgccgtctgcccttgcgcagaacaacgcgccggtaaccttcaccgacccggactcgggcattaccttcaacacgtggggtctcgccgaggattctccccagactaagggcggtttcacttttggtgttgctctgccctctgatgccctcacgacagacgccaaggagttcatcggttacttgaaatgcgcgaggaacgatgagagcggttggtgcggtgtctccctgggcggccccatgaccaactcgctcctcatcgcggcctggccccacgaggacaccgtctacacctctctccgcttcgccaccggctatgccatgccggatgtctaccagggggacgccgagatcacccaggtctcctcctctgtcaactcgacgcacttcagcctcatcttcaggtgcgagaactgcctgcaatggagtcaaagcggcgccaccggcggtgcctccacctcgaacggcgtgttggtcctcggctgggtccaggcattcgccgaccccggcaacccgacctgccccgaccagatcaccctcgagcagcacgacaacggcatgggtatctggggtgcccagctcaactccgacgccgccagcccgtcctacaccgagtgggccgcccaggccaccaagaccgtcacgggtgactgcggcggtcccaccgagacctctgtcgtcggtgtccccgttccgacgggcgtctcgttcgattacatcgtcgtgggcggcggtgccggtggcatccccgccgccgacaagctcagcgaggccggcaagagtgtgctgctcatcgagaagggctttgcctcgaccgccaacaccggaggcactctcggccccgagtggctcgagggccacgaccttacccgctttgacgtgccgggtctgtgcaaccagatctgggttgactccaaggggatcgcttgcgaggataccgaccagatggctggctgtgtcctcggcggcggtaccgccgtgaatgccggcctgtggttcaagccctactcgctcgactgggactacctcttccctagtggttggaagtacaaagacgtccagccggccatcaaccgcgccctctcgcgcatcccgggcaccgatgctccctcgaccgacggcaagcgctactaccaacagggcttcgacgtcctctccaagggcctggccggcggcggctggacctcggtcacggccaataacgcgccagacaagaagaaccgcaccttctcccatgcccccttcatgttcgccggcggcgagcgcaacggcccgctgggcacctacttccagaccgccaagaagcgcagcaacttcaagctctggctcaacacgtcggtcaagcgcgtcatccgccagggcggccacatcaccggcgtcgaggtcgagccgttccgcgacggcggttaccaaggcatcgtccccgtcaccaaggttacgggccgcgtcatcctctctgccggtacctttggcagtgcaaagatcctgctgaggagcggtatcggtccgaacgatcagctgcaggttgtcgcggcctcggagaaggatggccctaccatgatcagcaactcgtcctggatcaacctgcctgtcggctacaacctggatgaccacctcaacaccgacactgtcatctcccaccccgacgtcgtgttctacgacttctacgaggcgtgggacaatcccatccagtctgacaaggacagctacctcaactcgcgcacgggcatcctcgcccaagccgctcccaacattgggcctatgttctgggaagagatcaagggtgcggacggcattgttcgccagctccagtggactgcccgtgtcgagggcagcctgggtgcccccaacggcaagaccatgaccatgtcgcagtacctcggtcgtggtgccacctcgcgcggccgcatgaccatcaccccgtccctgacaactgtcgtctcggacgtgccctacctcaaggaccccaacgacaaggaggccgtcatccagggcatcatcaacctgcagaacgccctcaagaacgtcgccaacctgacctggctcttccccaactcgaccatcacgccgcgccaatacgttgacagcatggtcgtctccccgagcaaccggcgctccaaccactggatgggcaccaacaagatcggcaccgacgacgggcgcaagggcggctccgccgtcgtcgacctcaacaccaaggtctacggcaccgacaacctcttcgtcatcgacgcctccatcttccccggcgtgcccaccaccaaccccacctcgtacatcgtgacggcgtcggagcacgcctcggcccgcatcctcgccctgcccgacctcacgcccgtccccaagtacgggcagtgcggcggccgcgaatggagcggcagcttcgtctgcgccgacggctccacgtgccagatgcagaacgagtggtactcgcagtgcttgtga  (SEQ ID NO: 2)MRTSSRLIGALAAALLPSALAQNNAPVTFTDPDSGITFNTWGLAEDSPQTKGGFTFGVALPSDALTTDAKEFIGYLKCARNDESGWCGVSLGGPMTNSLLIAAWPHEDTVYTSLRFATGYAMPDVYQGDAEITQVSSSVNSTHFSLIFRCENCLQWSQSGATGGASTSNGVLVLGWVQAFADPGNPTCPDQITLEQHDNGMGIWGAQLNSDAASPSYTEWAAQATKTVTGDCGGPTETSVVGVPVPTGVSFDYIVVGGGAGGIPAADKLSEAGKSVLLIEKGFASTANTGGTLGPEWLEGHDLTRFDVPGLCNQIWVDSKGIACEDTDQMAGCVLGGGTAVNAGLWFKPYSLDWDYLFPSGWKYKDVQPAINRALSRIPGTDAPSTDGKRYYQQGFDVLSKGLAGGGWTSVTANNAPDKKNRTFSHAPFMFAGGERNGPLGTYFQTAKKRSNFKLWLNTSVKRVIRQGGHITGVEVEPFRDGGYQGIVPVTKVTGRVILSAGTFGSAKILLRSGIGPNDQLQVVAASEKDGPTMISNSSWINLPVGYNLDDHLNTDTVISHPDVVFYDFYEAWDNPIQSDKDSYLNSRTGILAQAAPNIGPMFWEEIKGADGIVRQLQWTARVEGSLGAPNGKTMTMSQYLGRGATRGRMTITPSLTTVVSDVPYLKDPNDKEAVIQGIINLQNALKNVANLTWLFPNSTITPRQYVDSMVVSPSNRRSNHWMGTNKIGTDDGRKGGSAVVDLNTKVYGTDNLFVIDASIFPGVPTTNPTSYIVTASEHASARILALPDLTPVPKYGQCGGREWSGSFVCADGSTCQMQNEWYSQCL CDH2: (SEQ ID NO: 3)MKLLSRVGATALAATLSLQQCAAQMTEGTYTDEATGIQFKTWTASEGAPFTFGLTLPADALEKDATEYIGLLRCQITDPASPSWCGISHGQSGQMTQALLLVAWASEDTVYTSFRYATGYTLPGLYTGDAKLTQISSSVSEDSFEVLFRCENCFSWDQDGTKGNVSTSNGNLVLGRAAAKDGVTGPTCPDTAEFGFHDNGFGQWGAVLEGATSDSYEEWAKLATTTPETTCDGTGPGDKECVPAPEDTYDYIVVGAGAGGITVADKLSEAGHKVLLIEKGPPSTGLWNGTMKPEWLESTDLTRFDVPGLCNQIWVDSAGIACTDTDQMAGCVLGGGTAVNAGLWWKPHPADWDENFPEGWKSSDLADATERVFKRIPGTSHPSQDGKLYRQEGFEVISKGLANAGWKEISANEAPSEKNHTYAHTEFMFSGGERGGPLATYLASAAERSNFNLWLNTAVRRAVRSGSKVTGVELECLTDGGFSGTVNLNEGGGVIFSAGAFGSAKLLLRSGIGPEDQLEIVASSKDGETFTPKDEWINLPVGHNLIDHLNTDLIITHPDVVFYDFYAAWDEPITEDKEAYLNSRSGILAQAAPNIGPMMWDQVTPSDGITRQFQWTCRVEGDSSKTNSTHAMTLSQYLGRGVVSRGRMGITSGLSTTVAEHPYLHNNGDLEAVIQGIQNVVDALSQVADLEWVLPPPDGTVADYVNSLIVSPANRRANHWMGTAKLGTDDGRSGGTSVVDLDTKVYGTDNLFVVDASVFPGMSTGNPSAMIVIVAEQAAQRILA LRS CDH3:(SEQ ID NO: 4) MKFLRKSDRGSVLGSTLFSLAFLFYSPPTAAQSPPPDGAVYDYIVIGSGPGGGVVGANLAKAGYSVLLLEAGDDSPGAGFGVYTPTVTWDFYVKHYPEGDPRDNQYSHLTWLTPDGRYWVGQSGAPEGSRLLGVYYPRGATLGGSSMINAMVVWLPNDSDWDYHAEVTGDDSWRAENMHKIFQKIEKNNYLPRGTANHGFDGWFQTQMGTMVQTNRTGPLQGNGVMTTYAQDWNLTIPMSDLLIRDPNEIGPDRDQTSSIYGQVSHQFANGNRYSSRHYVQDAVSSGANLTVSLTSLATRILFDTVTEPDSPRATGVEYLFGKSLYRGDRRRADGAIGVNRTAVARREVIVSGGAFNSPQLLLLSGIGNATELEALGIPVIRDLPGVGRNLMDNQEMPIVGTGSPGGGPGAVAGVAMYKTRHPAHGERDMFLFGGPGFLFRGFWPNEAVHLPDEPAQPVYGVSMVKGSSVNNGGWVKLRSRDPTDTPEINFNHYAVGAEYDLEAVKDTVAWIRSVYRRVGIATVEPPCARGPDENGYCGEEDEAWIHKQTFGHHPTSTNKIGADDDPTAVLDSKFRVRGVRALRVVDASAFARIPGVFPVVSTFMISQKASDDILAELEAESR  CDH4: (SEQ ID NO: 5)MGFLAATLVSCAALASAASIPRPHAKRQVSQLRDDYDFVIVGGGTSGLTVADRLTEAFPAKNVLVIEYGDVHYAPGTFDPPTDWITPQPDAPPSWSFNSLPNPDMANTTAFVLAGQVVGGSSAVNGMFFDRASRHDYDAWTAVGGSGFEQSSHKWDWEGLFPFFQKSVTFTEPPADIVQKYHYTWDLSAYGNGSTPIYSSYPVFQWADQPLLNQAWQEMGINPVTECAGGDKEGVCWVPASQHPVTARRSHAGLGHYADVLPRANYDLLVQHQVVRVVFPNGPSHGPPLVEARSLADNHLFNVTVKGEVIISAGALHTPTVLQRSGIGPASFLDDAGIPVTLDLPGVGANLQDHCGPPVTWNYTEPYTGFFPLPSEMVNNATFKAEAITGFDEVPARGPYTLAGGNNAIFVSLPHLTADYGAITANIRAMVADGTAASYLAADVRTIPGMVAGYEAQLLVLADLLDNPEAPSLETPWATSEAPQTSSVLAFLLHPLSRGSVRLNLSDPLAQPVLDYRSGSNPVDIDLHLAHVRFLRGLLDTPTMQARGALETAPGSAVADSDEALGEYVRSHSTLSFMHPCCTAAMLPEDRGGVVGPDLKVHGAEGLRVVDMSVMPLLPGAHLSATAYAVGEKAADI IIQEWMDKEQ CDH5:(SEQ ID NO: 6) MELLRVSLAAVALSPLILFGVAAAHPTARSIARSTILDGADGLLPEYDYIIIGGGTSGLTVADRLTENRKRKFSRSPLPTSPARSSPAWCYSVLVLERGIFQNSSSVTTISGGSRGLFDPSLTFNINSVPQAGLDNRSIAVIGGLILGGSSGVNGLQVLRGQREDYDRWGSYFGPNSDWSWKGLLPYFKKAWNFHPPRPELVSQFDIKYDPSYWGNTSDVHASFPTTFWPVLKLEMAAFGDIPGVEYPPDSASGETGAYWHPASVDPATVLRSFARPAHWDNIEAARPNYHTLTGQRVLKVAFDGNRATSVVFVPANATDHSTARSVKAKKEIVLAAGAIHTPQILQASGVGPKQVLKEAGVPLVVDAPGVGSNFQDQPYVVAPTFNFTKFPFHPDFYDMILNQTFIAEAQAQFEKDRTGPHTIASGYCGSWLPLQIIAPNSWKDIARRYESQDPAAYLPAGTDETVIEGYRAQQKALARSMRSKQSAMYNFFLRGGYEEGSVVYLHPTSRGTVRINRSDPFFSPPEVDYRALSNPTDLEVLLEFTPFTRRYFLETRLKSLDPVELSPGANVTAPADIEAWLRSVMIPSSFHPIGTAAMLPRHLGGVVDENLLVYGVEGLSVVDASVMPDLPGSYTQ QTVYAIAEKAADLIKSRACDH6: (SEQ ID NO: 7) MQVASKLVAVTGGALALWLHPVAAQEGCTNISSTETYDYIVVGSGAGGIPVADRLSEAGHKVLLIEKGPPSTGRWGGIMKPEWLIGTNLTRFDVPGLCNQIWADPTGAICTDVDQMAGCMLGGGTAVNAGLWWKPHPADWDVNFPEGWHSEDMAEATERVFERIPGTITPSMDGKRYLSQGFDMLGGSLEAAGWEYLVPNEHPDRKNRTYGHSTFMYSGGERGGPLATYLVSAVQREGFTLWMNTTVTRIIREGGHATGVEVQCSNSEAGQAGIVPLTPKTGRVIVSAGAFGSAKLLFRSGIGPKDQLNIVKNSTDGPSMISEDQWIELPVGYNLNDHVGTDIEIAHPDVVFYDYYGAWDEPIVEDTERYVANRTGPLAQAAPNIGPIFWETIKGSDGVSRHLQWQARVEGKLNTSMTITQYLGTGSRSRGRMTITRRLNTVVSTPPYLRDEYDREAVIQGIANLRESLKGVANLTWITPPSNVTVEDFVDSIPATPARRCSNHWIGTAKIGLDDGREGGTSVVDLNTKVYGTDNIFVVDASIFPGHITGNPSAAIVIAAEYAAAKILALPAPEDAAS  CDH7: (SEQ ID NO: 8)MASVDLDQPFDYIVVGGGTAGLVVANRLSEDSNVRVLVVEAGADRNADPLVLTPGLVAGLYGKDEYDWNFSSPPQPTLNNRRINQARGKMLGGTSGLNFMMLLYPSKGNIDSWAALGNPSWNYDALAPYLRKFATVHPSPQSARDLLGLTYIDESLAAGDGPIQVSHTDGHNVTNKAWLETFASLGLEVSTDPRDGKALGAFQNHASIDPATHTRSFAGPAYYTPDVAKRPNLVVLTETLVARVLFDTAGGEGDAVATGVEIITKDGQKKQVSACGEVILAAGALQSPQILELSGVGGRELLEKHNIPVVVDNPNVGEHVQDHPIVCQSFEVADGVPSGDVLRDPNVLQAVVGMYQSGGGAGPLGQSVISVAYTPLVDGSGVVSAEAKAELLARHESSFSTAEGKVLRDLVESPSEATFEFLLFPSQVDIPENPTSMAQYITPVLPENYISVMTFIHQPFSRGKVHITSPDIRAAPLWDPRYNSDPLDLELLARGVQFVERIVDSATPFGRVLKQGGKRQPPLRADDLETAREIVRQRQISVFHVSGSCTMRPRDQGGVVDERLRVYGTRGLRVVDASVFPIEPVGNI QSVVYAVAERAADLIKEDRAKA

EXAMPLE 1 Fungal Strains and Methods

This Example describes the production of variants of fungal strain C1.

Strain Nomenclature

Strain CF-200 (UV18#100fΔalp1) is a derivative C1 strain. Strain CF-400is a derivative of C1 strain (“UV18#100fΔalp1Δpyr5”), further modifiedby deletion of cdh1, wherein cdh1 comprises the polynucleotide sequenceof SEQ ID NO:1. Cellulolytic enzymes from these strains were produced bysubmerged liquid culture fermentation using methods and a suitablefungal growth medium, as well-known in the art.

GOPOD Assay

The GOPOD assay kits (Sigma-Aldrich) used in these experiments tomeasure the amount of glucose produced. In these experiments, 10 ul oftest sample was added to 190 ul of the GOPOD assay mix provided in thekit. The reaction was allowed to shake for 30 min at 50° C. Absorbanceof the solution was measured at 510 nm to determine the amount ofglucose produced. The glucose concentration of the samples wascalculated in comparison with the glucose standards (0-150 g/L).

EXAMPLE 2 Purification of C1 CDH1

In this Example, 400 mL of C1 supernatant produced using the methods ofExample 1 were first concentrated to 140 mL using a rotary evaporator.Then, 63 mL of the concentrate was buffer-exchanged into 20 mM MOPSbuffer, pH 7.0, using 4 in-line Hi-Prep 26/10 desalting columns (GEHealthcare, 17-5087-02). The resulting buffer-exchanged supernatant(˜150g/L total protein) was loaded onto a column containing 500 mL DEAEFast Flow resin (GE Healthcare, 17-0709-01) pre-equilibrated with 20 mMpH7.0 MOPS buffer. The column was rinsed with 1 column volume (CV) of 20mM MOPS (pH7.0) and then a 0-300 mM sodium chloride gradient was runover 12 column volumes. Fractions were collected and analyzed by NuPage®Novex® Bis-tris SDS-PAGE gels (Invitrogen, NP0322BOX). The SDS-PAGEbands corresponding to the apparent molecular weight of CDH1 wereanalyzed by MS (performed by Alphalyse). The mass-mapping analysisconfirmed the presence of CDH1 in late-eluting fractions. Fractionscontaining CDH1, as demonstrated by SDS-PAGE gel, and confirmed by MSwere pooled and concentrated by ultrafiltration using Sartoriuscentrifugal 10 kDa filter (Sartorius-Stedim, VS2002). Then, 10 mL 500 mMpiperazine (pH 5.6) and 45 mL saturated ammonium sulfate were added to45 mL of the CDH1-containing pool and the resulting mixture was loadedonto a Phenyl FF (high sub) 16/10 column (GE Healthcare, 28-9365-45)pre-equilibrated with 1.6M ammonium sulfate in 50 mM piperazine, pH 5.6.A gradient of 1.6 M to 0 M ammonium sulfate in 50 mM piperazine, pH 5.6,was run over 30 CV. Fractions were collected and SDS-PAGE gel analysiswas performed on the selected fractions as described above, revealingthat CDH1 eluted in the final rinse step with approximately 80-90%purity.

CDH1 activity was measured using a DCPIP reduction assay similar to thatdescribed by Schou et. al. (See, Schou et al., Biochem J., 330:565-71[1998]). In a UV-transparent flat-bottom 96-well plate, 50 μLCDH1-containing fractions were added to 150 μL of a solution of 1.0 g/Lcellobiose and 100 μM DCPIP in 100 mM sodium acetate, pH 5.0. Sampleswere agitated briefly at room temperature and then the absorbance at 530nm (A₅₃₀) was measured for 10 minutes. C1 CDH1-containing fractionsdisplayed a rapid drop in absorbance at 530 nm DCPIP assays wereperformed using varying amounts of glucose or cellobiose with purifiedCDH1. Serial dilutions of cellobiose (1.0 g/L to 7.8 mg/L) and glucose(10 g/L to 78 mg/L) were prepared in a 96-well shallow-well plate. Then,20 μL glucose and cellobiose standards were added to 160 μL/well 200 mMDCPIP (in 100 mM pH 5.0 sodium acetate). Reactions were initiated byaddition of 20 μL CDH1 solution. Absorbance at 530 nm was monitored for30 minutes. Comparisons of the rates of decrease in absorbance at 530 nmindicate that C1 CDH1 is approximately 10-fold more active on cellobiosethan glucose.

EXAMPLE 3 Making of CDH1 Split Marker Deletion Constructs

Genomic DNA was isolated from the C1 strain using standard procedures.Briefly, hyphal inoculum was seeded into a growth medium and allowed togrow for 72 hours at 35° C. The mycelial mat was collected bycentrifugation, washed, and 50 μL DNA extraction buffer (200 mM TRIS, pH8.0; 250 mM NaCl; 125 mM EDTA; 0.5% SDS) was added. The mycelia wereground with a conical grinder, re-extracted with 250 μL extractionbuffer, and the suspension was centrifuged. The supernatant wastransferred to a new tube containing 300 μL isopropanol. DNA wascollected by centrifugation, washed twice with 70% ethanol, and dilutedin 100 μL of water.

Genomic DNA fragments flanking the cdh1 gene were cloned using primerscf09067 and cf09068 (cdh1 upstream homology) and primers cf09069 andcf09070 (cdh1 downstream homology). PCR reactions were performed byusing the GoTaq® polymerase (Promega) following the manufacturer'sinstructions using 0.2 uM of each primer. The amplification conditionswere 95° C. for 2 minutes, 35 cycles of 95° C. for 30 seconds, 55° C.for 30 seconds (for upstream homology) or 53° C. for 30 seconds (fordownstream homology), 72° C. for 1 minute and final extension at 72° C.for 5 minutes. The pyr5 gene was PCR amplified as a split marker from avector using primers cf09024 and cf09025 (for the 5′ portion of thegene) and cf09026 and cf09027 (for the 3′ portion of the gene). PCRreactions were performed using the GoTaq® polymerase (Promega) followingthe manufacturer's instructions using 0.2 uM of each primer. Theamplification conditions were 95° C. for 2 minutes, 35 cycles of 95° C.for 30 seconds, 53° C. for 30 seconds, 72° C. for 1 minute and finalextension at 72° C. for 5 minutes. The primers used are shown in Table3-1. In separate strand overlap extension reactions (See, Horton et al.,Meth. Enzymol., 217:270-279 [1993]), the PCR products resulting fromprimers cf09067 and cf09068 and primers cf09026 and cf09027 were fused,as were the PCR products resulting from primers cf09069 and cf09070, andprimers cf09024 and cf09025. PCR reactions were performed by usingFinnzymes' Phusion® DNA polymerase following the manufacturer'sinstructions including 3% DMSO and using 0.2 uM of each primer. Theamplification conditions were 98° C. for 1 minute, 35 cycles of 98° C.for 10 seconds, 62° C. for 20 seconds, 72° C. for 2 minutes and finalextension at 72° C. for 5 minutes. The strand overlap extension productswere used for cdh1 deletion.

TABLE 3-1 Primer Names and Sequences Primer SEQ ID Name Sequence (5′-3′)NO: cf09067 CACGCGGGGTTCTTTCTCCATCTC  9 cf09068TGAGGAAAACGCCGAGACTGAGCTCGACTCTG 10 CCGGCCTACCTACGA cf09069ATCAGTTGGGTGCACGAGTGGGTTTTGATGGG 11 GAGTTGAGTTTGTGAA cf09070GGATGGATGAGGTTGTTTTTGAGC 12 cf09024 AACCCACTCGTGCACCCAACTGAT 13 cf09025GACCACGATGCCGGCTACGATACC 14 cf09026 ACATGGCCCCACTCGCTTCTTACA 15

EXAMPLE 4 Transformation Method

C1 cells and derivative strains were inoculated into 100 mL growthmedium in a 500 mL Erlenmeyer flask using 10⁶ spores/mL. The culture wasincubated for 48 hours at 35° C., 250 rpm. To harvest the mycelia, theculture was filtered over a sterile Myracloth filter (CalBiochem) andwashed with 100 mL 1700 mosmol NaCl/CaCl₂ solution (0.6 M NaCl, 0.27 MCaCl₂*H₂O). The washed mycelia were transferred into a 50 mL tube andweighed. Caylase (20 mg/gram mycelia; Cayla) was dissolved in 1700mosmol NaCl/CaCl₂ and UV-sterilized for 90 sec. Then, 3 mL of sterileCaylase solution was added into the tube containing washed mycelia andmixed. Then, 15 mL of 1700 mosmol NaCl/CaCl₂ solution was added into thetube and mixed. The mycelium/Caylase suspension was incubated at 30° C.,70 rpm for 2 hours. Protoplasts were harvested by filtering through asterile Myracloth filter into a sterile 50 mL tube. 25 mL cold STC (1.2M sorbitol, 50 mM CaCl₂*H₂O, 35 mM NaCl, 10 mM Tris-HCl) was added tothe flow through and spun down at 2720 rpm for 10 min at 4° C. Thepellet was resuspended in 50 mL STC and centrifuged again. After thewashing steps, the pellet was resuspended in 1 mL STC.

Then, 2 μg DNA of each strand overlap extension product was pipettedinto the bottom of a 15 mL sterile tube and 1 μL aurintricarboxylic acidand 100 μL of the protoplast suspension were added. The contents weremixed and the protoplasts were incubated with the DNA at roomtemperature for 25 min Then, 1.7 mL PEG4000 solution (60% PEG4000;polyethylene glycol, average molecular weight 4000 daltons), 50 mMCaCl₂.H₂O, 35 mM NaCl, 10 mM Tris-HCl) was added and mixed thoroughly.The solution was kept at room temperature for 20 min The tube was filledwith STC, mixed and centrifuged at 2500 rpm for 10 min at 4° C. The STCwas poured off and the pellet was resuspended in the remaining STC andplated on minimal selective media plates. The plates were incubated for5 days at 35° C. Colonies were restreaked and checked for the deletionof cdh1; colonies with this deletion were designated as strain “CF-400”.

EXAMPLE 5 Confirmation of CDH1 Deletion

Genomic DNA was prepared as described in Example 3. PCR reactions wereperformed by using the GoTaq® polymerase (Promega) following themanufacturer's instructions using 0.2 uM of each primer (primers cf09112and cf09113). The amplification conditions were 95° C. for 2 minutes, 35cycles of 95° C. for 30 seconds, 54° C. for 30 seconds, 72° C. for 30seconds and final extension at 72° C. for 5 minutes. PCR was alsoconducted using primers cf09110 and cf09111 and GoTaq® polymerase(Promega) following the manufacturer's instructions using 0.2 uM of eachprimer. The amplification conditions were 95° C. for 2 minutes, 35cycles of 95° C. for 30 seconds, 55.4° C. for 30 seconds, 72° C. for 30seconds and final extension at 72° C. for 5 minutes). These primers wereused in separate PCR reactions to confirm absence of the cdh1 gene.Primers cf09181 and cf09091 were used in PCR to confirm proper junctionstructure and targeting of the pyr5 marker construct (See, Table 5-1).The PCR reaction was performed by using the GoTaq® polymerase (Promega)following the manufacturer's instructions using 0.2 uM of each primer.The amplification conditions were 95° C. for 2 minutes, 35 cycles of 95°C. for 30 seconds, 54.4° C. for 30 seconds, 72° C. for 3 minutes 30seconds, and final extension at 72° C. for 5 minutes. PCR products wererun on an agarose gel to confirm a banding pattern indicative of cdh1deletion.

TABLE 5-1 Primer Names and Sequences Primer name Sequence (5′-3′)SEQ ID NO: cf09110 AAGCGTGCCGATTTTCCTGATTTC 16 cf09111GCATTTCTGGGGCGGTTAGCA 17 cf09112 TCATCGACGCCTCCATCTTCC 18 cf09113TTTCGGTTGTCGTGTTTCCATTAT 19 cf09181 GGAGATCCTGGAGGATTTCC 20 cf09091CAGGCGGTGTGCGTTATCAAAA 21

A colorimetric dichlorophenolindophenol (DCPIP) assay was used to testfor deletion of cdh1 in CF-400. Deletion of cdh1 was determined byobserving a decreased ability to reduce the DCPIP substrate compared toa parent strain. Cells of the parental C1 strain and putative cdh1delete strain were grown and the supernatants tested for DCPIP activity.In these tests, 160 μL of freshly made DCPIP reagent solution (0.2 mMDCPIP in 100 mM sodium acetate, pH 5.0), 20 μL cellobiose solution (1g/L cellobiose in deionized water), and 20 mLs of undiluted cellsupernatant were combined in microtiter plates. The absorbance of thesolution was immediately measured over time at 530 nm in kinetic modefor 30 minutes to track loss of absorbance as a result of DCPIPreduction. Supernatant from strains displaying decreased ability toreduce the DCPIP substrate were run on SDS-PAGE to confirm the absenceof CDH1. Proteins from culture supernatants of submerged liquid culturefermentations of CF-400 and the untransformed parent were separated bySDS-PAGE using standard protocols. The proteins were visualized bystaining with Simply Blue Safe Stain (Invitrogen), as per manufacturer'sinstructions. The Cdhl protein was observed as a ˜90 kD band in theuntransformed parent but was absent in CF-400.

EXAMPLE 6 Hydrolysis of Corn Stover

In these experiments, acid pretreated corn stover (NREL) was pH adjustedto 5.0 with aqueous ammonium hydroxide. The material was 41.3% solids,with a moisture content of 58.7%. The glucan content in the solids was40.7%. The acid pretreated corn stover was loaded into a 96-well plateand diluted with sodium acetate buffer to an average volume of 110 μLper well with 128 mM sodium acetate, at pH 5. The total solids loadingwere 24.7% in all experiments, and the concentration of glucan was 100 gglucan/kg reaction. CF-200 and CF-400 enzyme supernatants were used at 3g cellulase/kg reaction. A set of wells in the 96-well plate was alsorun wherein water was used in place of enzyme to serve as a control, dueto the presence of free glucose in the substrate. The level of thiscontrol was subtracted from the final measured glucose concentration.The plate was sealed once all reaction components were added and placedin a shaker at 55° C. rotating at 950 rpm for 73 hours. At the end ofreaction, the plate was allowed to cool. Samples were withdrawn, dilutedand subsequently analyzed by GO assay kit (Sigma) to determine glucoseproduction. The results are provided in FIG. 2. As indicated, the CF-200supernatant generated 52.1 g/L glucose, while CF-400 supernatantgenerated 69.4 g/L glucose. CF-400 supernatant exhibited highersaccharification performance, indicating that deletion of cdh1 genereduces formation of the gluconate from glucose during thesaccharification reaction.

While particular embodiments of the present invention have beenillustrated and described, it will be apparent to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the present invention. Therefore,it is intended that the present invention encompass all such changes andmodifications with the scope of the present invention.

The present invention has been described broadly and generically herein.Each of the narrower species and subgeneric groupings falling within thegeneric disclosure also form part(s) of the invention. The inventiondescribed herein suitably may be practiced in the absence of any elementor elements, limitation or limitations which is/are not specificallydisclosed herein. The terms and expressions which have been employed areused as terms of description and not of limitation. There is nointention that in the use of such terms and expressions, of excludingany equivalents of the features described and/or shown or portionsthereof, but it is recognized that various modifications are possiblewithin the scope of the claimed invention. Thus, it should be understoodthat although the present invention has been specifically disclosed bysome preferred embodiments and optional features, modification andvariation of the concepts herein disclosed may be utilized by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

What is claimed is:
 1. An enzyme mixture comprising two or morecellulose hydrolyzing enzymes, wherein at least one of the two or morecellulose hydrolyzing enzymes is expressed by a fungal cell that hasbeen genetically modified to reduce the amount of endogenous glucoseand/or cellobiose oxidizing enzyme activity that is produced by thefungal cell.
 2. An enzyme mixture comprising two or more cellulosehydrolyzing enzymes, wherein at least one of the two or more cellulosehydrolyzing enzymes is expressed by a fungal cell that has beengenetically modified to reduce the amount of endogenous glucose and/orcellobiose oxidizing enzyme activity that is secreted by the fungalcell.
 3. The enzyme mixture of claim 1, comprising two or more cellulosehydrolyzing enzymes, wherein at least one of the two or more cellulosehydrolyzing enzymes is expressed by a fungal cell that has beengenetically modified to reduce the amount of endogenous glucose and/orcellobiose oxidizing enzyme activity that is secreted by the fungal celland further to increase the expression of at least one saccharidehydrolyzing enzyme.
 4. The enzyme mixture of claim 1, wherein the enzymemixture is a cell-free mixture.
 5. The enzyme mixture of claim 1,wherein a substrate of the enzyme mixture comprises pretreatedlignocellulose.
 6. The enzyme mixture of claim 1, wherein the enzymemixture comprises at least one cellulase enzyme selected fromendoglucanases (EGs), beta-glucosidases (BGLs), Type 1cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), and/orglycoside hydrolase 61s (GH61s), and/or variants of said cellulaseenzyme.
 7. The enzyme mixture of claim 6, wherein the enzyme mixturecomprises at least one beta-glucosidase.
 8. The enzyme mixture of claim1, further comprising at least one cellobiose dehydrogenase.
 9. Theenzyme mixture of claim 8, wherein said celliobiose dehydrogenase isCDH1 and/or CDH2.
 10. The enzyme mixture of claim 1, further comprisingat least one cellulase enzyme and/or at least one additional enzyme. 11.The enzyme mixture of claim 1, wherein the enzyme mixture has beensubjected to a purification process to selectively remove one or moreglucose and/or cellobiose oxidizing enzymes from the enzyme mixture. 12.The enzyme mixture of claim 11, wherein the purification processcomprises selective precipitation to separate the glucose and/orcellobiose oxidizing enzymes from other enzymes present in the enzymemixture.
 13. The enzyme mixture of claim 1, wherein the enzyme mixturecomprises an inhibitor of one or more glucose and/or cellobioseoxidizing enzymes.